Humanized or chimeric cd3 antibodies

ABSTRACT

The present invention relates to humanized or chimeric antibodies binding CD3. It furthermore relates to bispecific antibodies, compositions, pharmaceutical compositions, use of said antibodies in the treatment of a disease, and method of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 15/744,317 filed Jan. 12, 2018, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP2016/066845, filed Jul. 14, 2016, which claims priority to International Application No. PCT/EP2016/050296, filed Jan. 8, 2016, which claims priority to Danish Patent Application Nos. PA 2015 00413 filed Jul. 15, 2015, PA 2015 00414 filed Jul. 15, 2015, and PA 2015 00416 filed Jul. 16, 2015. The entire contents of these applications are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 9, 2022, is named GMI-147USFCDV_SequenceListing_20220509.txt and is 298,446 bytes in size.

FIELD OF INVENTION

The present invention relates to a humanized or chimeric antibody binding to human CD3, compositions comprising said humanized or chimeric antibody, and use of said humanized or chimeric antibodies in treatment of a disease.

BACKGROUND

The Cluster of Differentiation 3 (CD3) has been known for many years and therefore has been subject of interest in many aspects. Specifically antibodies raised against CD3 or the T cell Receptor Complex, which CD3 is part of, are known. An in vitro characterization of recombinant chimeric CD3 isotype variants as well as a number of humanized OKT3 effector function variant antibodies has been described [1].

-   -   CD3 antibodies, e.g. muromonab-CD3, have been widely used in the         treatment of acute allograft rejection. In addition, treatment         with the anti-CD3 monoclonal antibody hOKT3gamma1(Ala-Ala)         results in improved C-peptide responses and clinical parameters         for at least 2 years after onset of type 1 diabetes in absence         of continued immunosuppressive medications [2].     -   A promising approach to improve targeted antibody therapy is by         delivering cytotoxic cells specifically to the         antigen-expressing cancer cells. This concept of using T cells         for efficient killing of tumor cells has been described [3].         However, initial clinical studies were rather disappointing         mainly due to low efficacy, severe adverse effects (cytokine         storm) and immunogenicity of the bispecific antibodies [4].         Advances in the design and application of bispecific antibodies         have partially overcome the initial barrier of cytokine storm         and improved clinical effectiveness without dose-limiting         toxicities [5].     -   For example, certain bispecific antibodies targeting with one         arm the antigen on the tumor cell and with the other arm for         instance CD3 on T cells, and containing an active Fc fragment         providing Fc receptor binding have been shown to induce tumor         cell killing. Upon binding, a complex of T cells, tumor cells         and effector cells that bind the antibody Fc region is         potentially formed, leading to killing of the tumor cells [4].         Catumaxomab consists of a mouseIgG2a/ratIgG2b heavy chain         heterodimer and has been found successful for the treatment of         cancer-associated ascites after intraperitoneal application [6].         However, the mouse/rat hybrid is immunogenic [7] and cannot be         applied for long-term treatment in humans. Frequent         treatment-related adverse events attributed to catumaxomab         included cytokine-release-related symptoms (i.e. pyrexia,         nausea, vomiting, chills, tachycardia and hypotension) [8]-[9],         which relate the potent polyclonal T cell activation by         catumaxomab due to its active Fc fragment. Another antibody is         ertumaxomab (HER2×CD3), which induces cytotoxicity in cell lines         with HER2 expression. Ertumaxomab has been in Phase II clinical         development for metastatic breast cancer [10]-[11].     -   Efficacy of CD3 bispecific antibodies and other CD3 bispecific         antibody-based formats is dependent on several properties of         bispecific antibodies, such as the affinity of the CD3 arm         and/or the affinity to the target of the second arm and the         target copy number on target cells. Some CD3 bispecific         antibodies show high efficacy when the CD3 affinity is low         (EpCamxCD3—Bortoletto 2002 PMID 12385030, MT103/Blinatumomab vs         TandAb—Molhoj 2007 PMID 17083975), while other CD3 bispecifics         demonstrate high efficacy using a high CD3 affinity (Reusch         2015, Mabs, PMID 25875246). In some cases high CD3 affinity is         required, for example when arming ex vivo expanded activated T         cells from patients with a bispecific antibody comprised of an         anti-CD3 targeting arm and a second arm directed at a selected         tumor-associated antigen. In the latter case, CD3 affinity         should be high to retain the interaction with the expanded T         cell when the product is infused back into the patient to         mediate cytolysis of tumor cells (Reusch 2006 Clin Cancer Res         PMID 16397041). However, high affinity antibodies to CD3, in         contrast to low affinity ligands, are much less effective in TCR         triggering at low copy number, since they display a         stoichiometry of ˜1:1 and a linear dose-response curve,         indicative of a single cycle rather than a serial triggering         mode of T cell response (Viola 1996 Science, PMID 8658175). In         other words, low affinity of CD3 arm can allow T cells to         flexibly move from one target and/or target cell to the other         (Hoffman 2005, PMID: 15688411).     -   Low CD3 affinity can potentially prevent the biased localization         of the bispecific antibody to T cells (due to first encounter in         circulation) and thus improve biodistribution and minimize         interference with normal T cell immune responses. Depending on         the target of the second arm and target copy number, the         indication and/or administration route, desirable CD3 affinity         can be customized to enhance a product's maximum efficacy. A         panel of CD3 variants covering a range of CD3 affinity can be         essential to suit these specific tailor-made needs per antibody         product.     -   CD3 antibodies cross-reactive to cynomolgus and/or rhesus monkey         CD3 have been described [12]-[13], however, further improvements         for such cross-reactive antibodies are needed.

SUMMARY OF INVENTION

The object of the present invention is to provide humanized or chimeric CD3 antibodies with an optimized affinity to CD3. Thus it is an object of the present invention to provide humanized or chimeric CD3 antibodies which are optimized compared to a reference antibody such as an antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. Hence, such antibodies may have a reduced or increased affinity to CD3 compared to a reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the present invention to provide antibodies with a lower binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. The inventors found that antibodies with a reduced binding affinity to the CD3 peptide as set forth in SEQ ID NO: 402 compared to a reference antibody having the VH region sequence set forth in SEQ ID NO: 4 maintain the same or similar cytotoxic activity in vitro and in vivo compared to the reference antibody. Another object of the present invention is to provide CD3 antibodies with reduced binding affinity to CD3 compared to a reference antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8, but retaining the same cytolytic activity as the reference antibody. It is yet another object of the present invention to provide antibodies with a higher binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8.

The present invention provides in one aspect a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises a mutation in one of the three CDR sequences of a reference antibody having the VH CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3, which mutation is in one of the positions selected from the group consisting of: T31M, T31P, N57, H101, G105, S110 and Y114, wherein the positions are numbered according to the reference sequence of the SEQ ID NO: 4. The amino acids in SEQ ID NO: 4 are numbered according to a direct numerical numbering scheme from the first amino acid to number 125 in the direction from N-terminus to the C-terminus. The numerical numbering of positions corresponding to SEQ ID NO:4 is illustrated in FIG. 2 . Further, the VH CDR regions have been annotated according to the IMGT definitions.

In one embodiment of the invention, the antibody has a reduced or increased binding affinity to human CD3 compared to the reference antibody having the VH CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3.

In some embodiments of the invention, an antibody with reduced binding affinity to a human CD3 molecule, such as a CD3 peptide e.g. SEQ ID NO:402, compared to a reference antibody may maintain the same cytolytic activity against a target cell as the reference antibody.

In one embodiment of the invention the antibody comprises a mutation in the position corresponding to N57 of SEQ ID NO: 4. In one embodiment the mutation is N57E.

In one embodiment of the invention the antibody comprises a mutation in the position corresponding to H101G of SEQ ID NO: 4. In one embodiment the mutation is H101G or H101N.

In one embodiment of the invention the antibody comprises a mutation in the position corresponding to G105 of SEQ ID NO: 4. In one embodiment the mutation is G105P.

In one embodiment of the invention the antibody comprises a mutation in the position corresponding to Y114 of SEQ ID NO: 4. In one embodiment the mutation is Y114M, Y114R or Y114V.

In one embodiment, the present invention provides a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 12, 2,         3;     -   b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 14, 2,         3;     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 16, 2,         3;     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 18, 2,         3;     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 20, 2,         3;     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 22, 2,         3;     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 24, 2,         3;     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 26, 2,         3;     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 28, 2,         3;     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 30, 2,         3;     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 32, 2,         3;     -   l) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 34, 2,         3;     -   m) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 36, 2,         3;     -   n) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 38, 2,         3;     -   o) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 40, 2,         3;     -   p) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 42, 2,         3;     -   q) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 44, 2,         3;     -   r) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 46, 2,         3;     -   s) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 48, 2,         3;     -   t) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 50, 2,         3;     -   u) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 52, 2,         3;     -   v) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3;     -   w) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 56, 2,         3;     -   x) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2,         3;     -   y) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 60, 2,         3;     -   z) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 62, 2,         3;     -   aa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 64, 2,         3;     -   bb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 66, 2,         3;     -   cc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 68, 2,         3;     -   dd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 70, 2,         3;     -   ee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 72, 2,         3;     -   ff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 74, 2,         3;     -   gg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 76, 2,         3;     -   hh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 78, 2,         3;     -   ii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 80, 2,         3;     -   jj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 82, 2,         3;     -   kk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 84, 2,         3;     -   ll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 86, 2,         3;     -   mm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 88, 2,         3;     -   nn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 90, 2,         3;     -   oo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 92, 2,         3;     -   pp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 94, 2,         3;     -   qq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 96, 2,         3;     -   rr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 98, 2,         3;     -   ss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         100, 3;     -   tt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         102, 3;     -   uu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         104, 3;     -   vv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         106, 3;     -   ww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         108, 3;     -   xx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         110, 3;     -   yy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         112, 3;     -   zz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         114, 3;     -   aaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         116, 3;     -   bbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         118, 3;     -   ccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         120, 3;     -   ddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         122, 3;     -   eee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         124, 3;     -   fff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         126, 3;     -   ggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         128, 3;     -   hhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         130, 3;     -   iii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         132, 3;     -   jjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         134, 3;     -   kkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         136, 3;     -   lll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         138, 3;     -   mmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         140, 3;     -   nnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         142, 3;     -   ooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         144, 3;     -   ppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         146, 3;     -   qqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         148, 3;     -   rrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         150, 3;     -   sss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         152, 3;     -   ttt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         154, 3;     -   uuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         156, 3;     -   vvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         158, 3;     -   www) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         160, 3;     -   xxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         162, 3;     -   yyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         164, 3;     -   zzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         166, 3;     -   aaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         168, 3;     -   bbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 170;     -   cccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 172;     -   dddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 174;     -   eeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 176;     -   ffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 178;     -   gggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 180;     -   hhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 182;     -   iiii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 184; CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 186;     -   kkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 188;     -   llll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 190;     -   mmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 192;     -   nnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 194;     -   oooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 196;     -   pppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 198;     -   qqqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 200;     -   rrrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 202;     -   ssss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 204;     -   tttt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 206;     -   uuuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 208;     -   vvvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 210;     -   wwww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 212;     -   xxxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 214;     -   yyyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 216;     -   zzzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 218;     -   aaaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 220;     -   bbbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 222;     -   ccccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 224;     -   ddddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 226;     -   eeeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 228;     -   fffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 230;     -   ggggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 232;     -   hhhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 234;     -   iiiii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 236;     -   jjjjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 238;     -   kkkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 240;     -   lllll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 242;     -   mmmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 244;     -   nnnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 246;     -   ooooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 248;     -   ppppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 250;     -   qqqqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 252;     -   rrrrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 254;     -   sssss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 256;     -   ttttt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 258;     -   uuuuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 260;     -   vvvvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 262;     -   wwwww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 264;     -   xxxxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 266;     -   yyyyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 268;     -   zzzzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 270;     -   aaaaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 272;     -   bbbbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 274;     -   cccccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 276;     -   dddddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 278;     -   eeeeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 280;     -   ffffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 282;     -   gggggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 284;     -   hhhhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 286;     -   iiiiii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 288;     -   jjjjjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 290;     -   kkkkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 292;     -   llllll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 294;     -   mmmmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 296;     -   nnnnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 298 and     -   oooooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 300.

That is, the inventors of the present invention in a first aspect of the invention found that humanized or chimeric antibodies of said sequences had an optimized binding affinity to a CD3 peptide SEQ ID NO: 402 compared to a reference antibody such as an antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. The reference antibody specified by SEQ ID NO:4 and the VL sequence SEQ ID NO:8 has a binding affinity to the CD3 peptide of SEQ ID NO:402 of 1.5×10⁻⁸ M as illustrated by example 7. In some embodiments of the present invention the antibodies have a lower binding affinity to the CD3 peptide of SEQ ID NO:402 than 1.5×10⁻⁸ M such as a binding affinity from 1.6×10⁻⁸ M to 9.9×10⁻⁸ M or such as a binding affinity from 1.0×10⁻⁷ to 9.9×10⁻⁷ M when determined by Bio-Layer Interferometry as described in Table 6 in example 7. In some embodiments of the present invention the antibodies have a higher binding affinity to CD3 peptide of SEQ ID NO: 402 than 1.5×10⁻⁸ M, such as from 1.4×10⁻⁸ to 1.0×10⁻⁸ M, such as 9.9×10⁻⁹ to 1×10⁻⁹ M or such as 9.9×10⁻⁹ to 1×10⁻⁹ M. The binding affinity corresponds to the K_(D) value.

In one aspect of the present invention, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequence set forth in SEQ ID NO: 58, 2, 3         [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114M];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R];     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V] and     -   l) CDR1, CDR2 and CDR3 sequences having at least 90% or at least         95% amino acid sequence identity, in total across the three CDR         sequences, to any one of the three CDR sequences as set forth         in a) to k), provided that the CDR1, CDR2 and CDR3 sequences do         not have the sequences as set forth in SEQ ID NO: 1, 2, 3.

In another aspect, the present invention relates to a humanized or chimeric antibody, wherein said binding region comprises a variable light chain (VL) region, wherein said VL region comprises CDR1, CDR2, and CDR3 regions having the CDR as set forth in SEQ ID NO: 6, GTN, 7.

In a further aspect, the present invention relates to a method of reducing the binding affinity of an antibody binding to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2, 3, which method comprises introducing a mutation in one of the three CDR sequences of the said reference antibody selected from a mutation in one of the positions selected from the group of T31M, T31P, N57, H101, S110 and Y114, wherein the positions are numbered according to the reference sequence of the SEQ ID NO: 4.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the present invention the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In a further embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 region selected from H101G, H101N, G105P, S110A, S110G, Y114M, Y114R or Y114V.

In one embodiment CD3 is human CD3 epsilon.

In another aspect, the present invention relates to a bispecific antibody comprising a first binding region of an antibody according to the invention, and a second binding region which binds a different target than said first antigen binding region.

In another aspect, the present invention relates to a nucleic acid construct encoding one or more amino acid sequences according to the invention.

In another aspect, the present invention relates to an expression vector comprising (i) a nucleic acid sequence encoding a heavy chain sequence of a humanized or chimeric antibody according to the invention, (ii) a nucleic acid sequence encoding a light chain sequence of a humanized or chimeric antibody according to the invention, or (iii) both (i) and (ii).

In another aspect, the present invention relates to a host cell comprising an expression vector according to the invention.

In another aspect, the present invention relates to a composition comprising the antibody or bispecific antibody according to the invention.

In another aspect, the present invention relates to a pharmaceutical composition comprising the antibody or bispecific antibody according to the invention and a pharmaceutical acceptable carrier.

In another aspect, the present invention relates to the antibody or bispecific antibody, the composition, or the pharmaceutical composition according to the invention for use as a medicament.

In another aspect, the present invention relates to the antibody or bispecific antibody, the composition, or the pharmaceutical composition according to the invention for use in the treatment of a disease.

In another aspect, the present invention relates to a method of treatment of a disease comprising administering the antibody or bispecific antibody, the composition, or the pharmaceutical composition according to the invention, to a subject in need thereof.

In another aspect, the present invention relates to a method of administering the antibody or bispecific antibody, wherein the antibody or bispecific antibody is administered by subcutaneous or local administration.

In one aspect, the present invention relates to a method of diagnosing a disease characterized by involvement or accumulation of CD3-expressing cells, comprising administering the humanized or chimeric antibody, the composition or the pharmaceutical composition according to the invention to a subject, optionally wherein said humanized or chimeric antibody is labeled with a detectable agent.

In another aspect, the present invention relates to a method for producing an antibody or a bispecific antibody according to the invention, comprising the steps of a) culturing a host cell according to the invention, and b) purifying the antibody from the culture media.

In another aspect, the present invention relates to a diagnostic composition comprising an antibody or bispecific antibody according to any one of the embodiments as disclosed herein.

In one embodiment, the diagnostic composition is a companion diagnostic which is used to screen and select those patients who will benefit from treatment with the bispecific antibody.

In another aspect, the present invention relates to a method for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising the steps of a) contacting the sample with an antibody or bispecific antibody according to the invention, under conditions that allow for formation of a complex between the antibody or bispecific antibody and CD3, and b) analyzing whether a complex has been formed.

In another aspect, the present invention relates to a kit for detecting the presence of CD3 antigen, or a cell expressing CD3, in a sample comprising i) an antibody or bispecific antibody according to the invention, and ii) instructions for use of the kit.

In another aspect, the present invention relates to an anti-idiotypic antibody or a pair of anti-idiotypic antibodies which bind to an antibody according to the invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 : Heat plot with binding ratios of mutant versus wt UniTE-huCD3-H1L1-T41K molecules. Ratios above 1 indicate stronger binding than wt, while ratios below 1 indicate weaker binding than wt. Binding was determined on Freestyle 293-F cells transfected with CD3/TCR-LC13.

FIG. 2 : Alignment of selected CD3 affinity variants in the generated library with mutations in the VH. CDRs are underlined in the humanized wild type sequence (SEQ ID NO:4) HuCD3-H1. Highlighted amino acids are the substitutions.

FIG. 3 : T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibodies as determined by flow cytometry Affinity variants depicted cover a broad range of T cell binding capacity between the wild type response and undetectable response.

FIG. 4 : T cell binding curves of selected VH affinity variants of humanized CD3 (UniTE-huCD3-H1L1-T41K) antibodies as determined by flow cytometry showing a very low, undetectable T cell binding.

FIG. 5 : T cell binding curves of selected VH affinity variants of humanized CD3 (BisG1-huCD3-H1L1-X-FEAL/1014-Herceptin-FEAR) antibodies as determined by flow cytometry. Affinity variants depicted cover a broad range of T cell binding capacity between the wild type response and undetectable response.

FIGS. 6A-6C: Cytotoxicity of CD3 affinity variants on solid tumor cell lines measured by alamar blue assay. (FIG. 6A) NCI-N87 cells, Effector cells (T cells):Tumor cell (NCI-N87 cell) ratio=3:1, 48 hours of incubation, n=2 donors (FIG. 6B) SKOV3 cells, T cells:SKOV3 cell ratio=4:1, 48 hours of incubation, n=2 donors (FIG. 6C) MDA-MB-231 cells, T cells:MDA-MB-231 cell ratio=8:1, 48 hours of incubation, n=2 donors. The tested affinity variants depicted cover a broad range of cytotoxicity between wild type response and no observed cytotoxicity for all tested tumor cell lines.

FIG. 7 : Cytotoxicity of CD3 affinity variants on a hematological (Daudi) cell line measured by chromium release assay. T cell: Daudi cell) ratio=10:1, 24 hours of incubation, 1 donor. The tested affinity variants depicted cover a broad range of cytotoxicity between wild type response and no observed cytotoxicity for the tested tumor cell line.

FIGS. 8A-8D: Cytotoxicity of CD3×HER2 bispecific antibodies in a NCI-N87, human PBMC co-engraftment model in NOD-SCID mice. HLA-A-matched human unstimulated PBMCs, as a source of human T cells, were co-inoculated with NCI-N87 tumor cells in NOD-SCID mice at two different dose levels of CD3 affinity antibodies (0.5 and 0.05 mg/kg). Humanized WT CD3 (huCD3) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were tested. (FIG. 8A) Average tumor volume followed over time after treatment with 0.05 mg/kg of antibody (n=4 per group). (FIG. 8B) Average tumor volume followed over time after treatment with 0.5 mg/kg of antibody (n=4 per group). (FIG. 8C) Average tumor volume at day 44 after treatment with 0.05 mg/kg of antibody at day 0 (n=4 per group). (FIG. 8D) Average tumor volume at day 44 after treatment with 0.5 mg/kg of antibody at day 0 (n=4 per group). Statistics has been performed on data for C and D.

DETAILED DESCRIPTION

In one aspect, the present invention relates to a humanized or chimeric antibody binding to human CD3 with an optimized affinity to CD3. Thus it is an object of the present invention to provide humanized or chimeric CD3 antibodies which are optimized compared to a reference antibody such as the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the invention to provide antibodies with optimized in vivo efficacy compared to a reference antibody such as the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is a further object of the present invention to provide antibodies with a lower binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8. It is yet another object of the present invention to provide antibodies with a higher binding affinity to CD3 than the antibody specified by the VH sequence SEQ ID NO:4 and the VL sequence SEQ ID NO:8.

In one aspect the invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises a mutation in one of the three CDR sequences of a reference antibody having the CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3, which mutation is in one of the positions selected from the group consisting of: T31M, T31P, N57, H101, G105, S110 and Y114, wherein the positions are numbered according to the reference sequence of the SEQ ID NO: 4. The amino acids in SEQ ID NO: 4 are numbered according to a direct numerical numbering scheme from the first amino acid to number 125 in the direction from N-terminus to the C-terminus. The numerical numbering of positions corresponding to SEQ ID NO: 4 is illustrated in FIG. 2 . Further, The CDR regions have been annotated according to the IMGT definitions.

In one embodiment of the invention, the antibody has a reduced or increased binding affinity to human CD3 compared to the reference antibody having the VH CDR sequences set forth in CDR1 SEQ ID NO: 1, CDR2 SEQ ID NO: 2 and CDR3 SEQ ID NO: 3.

In some embodiments of the invention, an antibody with reduced binding affinity to a CD3 molecule, such a CD3 peptide e.g. SEQ ID NO:402, compared to a reference antibody may maintain the same cytolytic activity against a target cell as the reference antibody.

In one embodiment of the invention the antibody comprises a T31M or T31P mutation. Position T31 is in accordance to SEQ ID NO:4.

In one embodiment of the invention the antibody comprises a mutation in the position N57. Position N57 is in accordance to SEQ ID NO:4. In one embodiment the mutation is N57E.

In one embodiment of the invention the antibody comprises a mutation in the position H101. Position H101 is in accordance to SEQ ID NO: 4. In one embodiment the mutation is H101G or H101N.

In one embodiment of the invention the antibody comprises a mutation in the position G105. Position G105 is in accordance to SEQ ID NO: 4. In one embodiment the mutation is G105P.

In one embodiment of the invention the antibody comprises a mutation in the position Y114. Position Y114 is in accordance to of SEQ ID NO: 4. In one embodiment the mutation is Y114M, Y114R or Y114V.

The reference antibody specified by SEQ ID NO:4 and the VL sequence SEQ ID NO:8 has a binding affinity to the CD3 peptide of SEQ ID NO:402 corresponding to a K_(D) value of 1.5×10⁻⁸ M as illustrated by example 7.

In some embodiments of the present invention the antibodies have a lower binding affinity to the CD3 peptide of SEQ ID NO:402 than 1.5×10⁻⁸ M, such as a binding affinity from 1.6×10⁻⁸ M to 9.9×10⁻⁸ M or such as a binding affinity from 1.0×10⁻⁷ to 9.9×10⁻⁷ M when determined by Bio-Layer Interferometry as described in Example 7. In some embodiments of the present invention the antibodies have a higher binding affinity to the CD3 peptide of SEQ ID NO:402 than 1.5×10⁻⁸ M such as from 1.4×10⁻⁸ to 1.0×10⁻⁸ M, such as, such as such as 9.9×10⁻⁹ to 1.0×10⁻⁹ M.

In one embodiment, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 12, 2,         3;     -   b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 14, 2,         3;     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 16, 2,         3;     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 18, 2,         3;     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 20, 2,         3;     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 22, 2,         3;     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 24, 2,         3;     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 26, 2,         3;     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 28, 2,         3;     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 30, 2,         3;     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 32, 2,         3;     -   l) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 34, 2,         3;     -   m) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 36, 2,         3;     -   n) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 38, 2,         3;     -   o) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 40, 2,         3;     -   p) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 42, 2,         3;     -   q) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 44, 2,         3;     -   r) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 46, 2,         3;     -   s) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 48, 2,         3;     -   t) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 50, 2,         3;     -   u) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 52, 2,         3;     -   v) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3;     -   w) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 56, 2,         3;     -   x) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2,         3;     -   y) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 60, 2,         3;     -   z) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 62, 2,         3;     -   aa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 64, 2,         3;     -   bb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 66, 2,         3;     -   cc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 68, 2,         3;     -   dd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 70, 2,         3;     -   ee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 72, 2,         3;     -   ff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 74, 2,         3;     -   gg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 76, 2,         3;     -   hh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 78, 2,         3;     -   ii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 80, 2,         3;     -   jj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 82, 2,         3;     -   kk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 84, 2,         3;     -   ll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 86, 2,         3;     -   mm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 88, 2,         3;     -   nn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 90, 2,         3;     -   oo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 92, 2,         3;     -   pp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 94, 2,         3;     -   qq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 96, 2,         3;     -   rr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 98, 2,         3;     -   ss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         100, 3;     -   tt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         102, 3;     -   uu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         104, 3;     -   vv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         106, 3;     -   ww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         108, 3;     -   xx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         110, 3;     -   yy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         112, 3;     -   zz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         114, 3;     -   aaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         116, 3;     -   bbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         118, 3;     -   ccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         120, 3;     -   ddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         122, 3;     -   eee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         124, 3;     -   fff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         126, 3;     -   ggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         128, 3;     -   hhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         130, 3;     -   iii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         132, 3;     -   jjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         134, 3;     -   kkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         136, 3;     -   lll) CDR sequences set forth in SEQ ID NO: 1, 138, 3;     -   mmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         140, 3;     -   nnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         142, 3;     -   ooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         144, 3;     -   ppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         146, 3;     -   qqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         148, 3;     -   rrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         150, 3;     -   sss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         152, 3;     -   ttt) CDR sequences set forth in SEQ ID NO: 1, 154, 3;     -   uuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         156, 3;     -   vvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         158, 3;     -   www) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         160, 3;     -   xxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         162, 3;     -   yyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         164, 3;     -   zzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         166, 3;     -   aaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         168, 3;     -   bbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 170;     -   cccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 172;     -   dddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 174;     -   eeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 176;     -   ffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 178;     -   gggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 180;     -   hhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 182;     -   iiii) CDR sequences set forth in SEQ ID NO: 1, 2, 184;     -   jjjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 186;     -   kkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 188;     -   llll) CDR sequences set forth in SEQ ID NO: 1, 2, 190;     -   mmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 192;     -   nnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 194;     -   oooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 196;     -   pppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 198;     -   qqqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 200;     -   rrrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 202;     -   ssss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 204;     -   tttt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 206;     -   uuuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 208;     -   vvvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 210;     -   wwww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 212;     -   xxxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 214;     -   yyyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 216;     -   zzzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 218;     -   aaaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 220;     -   bbbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 222;     -   ccccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 224;     -   ddddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 226;     -   eeeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 228;     -   fffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 230;     -   ggggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 232;     -   hhhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 234;     -   iiiii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 236;     -   jjjjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 238;     -   kkkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 240; 11111) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID         NO: 1, 2, 242;     -   mmmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 244;     -   nnnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 246;     -   ooooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 248;     -   ppppp) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 250;     -   qqqqq) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 252;     -   rrrrr) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 254;     -   sssss) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 256;     -   ttttt) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 258;     -   uuuuu) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 260;     -   vvvvv) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 262;     -   wwwww) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 264;     -   xxxxx) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 266;     -   yyyyy) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 268;     -   zzzzz) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 270;     -   aaaaaa) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 272;     -   bbbbbb) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 274;     -   cccccc) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 276;     -   dddddd) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 278;     -   eeeeee) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 280;     -   ffffff) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 282;     -   gggggg) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 284;     -   hhhhhh) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 286;     -   iiiiii) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 288;     -   jjjjjj) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 290;     -   kkkkkk) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 292;     -   llllll) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 294;     -   mmmmmm) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 296;     -   nnnnnn) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 298 and     -   oooooo) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1,         2, 300.

In one embodiment, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequence set forth in SEQ ID NO: 58, 2, 3         [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114N1];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R];     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V] and     -   l) CDR1, CDR2 and CDR3 sequences having at least 90% or at least         95% amino acid sequence identity, in total across the three CDR         sequences, to any one of the three CDR sequences as set forth         in a) to k), provided that the CDR1, CDR2 and CDR3 sequences do         not have the sequences as set forth in SEQ ID NO: 1, 2, 3.

In one embodiment, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequence set forth in SEQ ID NO: 58, 2, 3         [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114N1];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R];     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V]; and         -   a. and     -   l) CDR1, CDR2 and CDR3 sequences as specified in a) to k) having         at most 5 further mutations or substitutions, at most 4 further         mutations or substitutions, at most 3 further mutations or         substitutions, at most 2 further mutations or substitutions, or         at most 1 further mutation or substitution, in total across the         three CDR sequences, and which mutations or substitutions         preferably do not modify the binding affinity to human CD3.

In one embodiment, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region), wherein said VH region comprises one of the VH sequences from the group consisting of;

-   -   a) a VH sequence as set forth in SEQ ID NO: 13;     -   b) a VH sequence as set forth in SEQ ID NO: 15;     -   c) a VH sequence as set forth in SEQ ID NO: 17;     -   d) a VH sequence as set forth in SEQ ID NO: 19;     -   e) a VH sequence as set forth in SEQ ID NO: 21;     -   f) a VH sequence as set forth in SEQ ID NO: 23;     -   g) a VH sequence as set forth in SEQ ID NO: 25;     -   h) a VH sequence as set forth in SEQ ID NO: 27;     -   i) a VH sequence as set forth in SEQ ID NO: 29;     -   j) a VH sequence as set forth in SEQ ID NO: 31;     -   k) a VH sequence as set forth in SEQ ID NO: 33;     -   l) a VH sequence as set forth in SEQ ID NO: 35;     -   m) a VH sequence as set forth in SEQ ID NO: 37;     -   n) a VH sequence as set forth in SEQ ID NO: 39;     -   o) a VH sequence as set forth in SEQ ID NO: 41;     -   p) a VH sequence as set forth in SEQ ID NO: 43;     -   q) a VH sequence as set forth in SEQ ID NO: 45;     -   r) a VH sequence as set forth in SEQ ID NO: 47;     -   s) a VH sequence as set forth in SEQ ID NO: 49;     -   t) a VH sequence as set forth in SEQ ID NO: 51;     -   u) a VH sequence as set forth in SEQ ID NO: 53;     -   v) a VH sequence as set forth in SEQ ID NO: 55;     -   w) a VH sequence as set forth in SEQ ID NO: 57;     -   x) a VH sequence as set forth in SEQ ID NO: 59;     -   y) a VH sequence as set forth in SEQ ID NO: 61;     -   z) a VH sequence as set forth in SEQ ID NO: 63;     -   aa) a VH sequence as set forth in SEQ ID NO: 65;     -   bb) a VH sequence as set forth in SEQ ID NO: 67;     -   cc) a VH sequence as set forth in SEQ ID NO: 69;     -   dd) a VH sequence as set forth in SEQ ID NO: 71;     -   ee) a VH sequence as set forth in SEQ ID NO: 73;     -   ff) a VH sequence as set forth in SEQ ID NO: 75;     -   gg) a VH sequence as set forth in SEQ ID NO: 77;     -   hh) a VH sequence as set forth in SEQ ID NO: 79;     -   ii) a VH sequence as set forth in SEQ ID NO: 81;     -   jj) a VH sequence as set forth in SEQ ID NO: 83;     -   kk) a VH sequence as set forth in SEQ ID NO: 85;     -   ll) a VH sequence as set forth in SEQ ID NO: 87;     -   mm) a VH sequence as set forth in SEQ ID NO: 89;     -   nn) a VH sequence as set forth in SEQ ID NO: 91;     -   oo) a VH sequence as set forth in SEQ ID NO: 93;     -   pp) a VH sequence as set forth in SEQ ID NO: 95;     -   qq) a VH sequence as set forth in SEQ ID NO: 97;     -   rr) a VH sequence as set forth in SEQ ID NO: 99;     -   ss) a VH sequence as set forth in SEQ ID NO: 101;     -   tt) a VH sequence as set forth in SEQ ID NO: 103;     -   uu) a VH sequence as set forth in SEQ ID NO: 105;     -   vv) a VH sequence as set forth in SEQ ID NO: 107;     -   ww) a VH sequence as set forth in SEQ ID NO: 109;     -   xx) a VH sequence as set forth in SEQ ID NO: 111;     -   yy) a VH sequence as set forth in SEQ ID NO: 113;     -   zz) a VH sequence as set forth in SEQ ID NO: 115;     -   aaa) a VH sequence as set forth in SEQ ID NO: 117;     -   bbb) a VH sequence as set forth in SEQ ID NO: 119;     -   ccc) a VH sequence as set forth in SEQ ID NO: 121;     -   ddd) a VH sequence as set forth in SEQ ID NO: 123;     -   eee) a VH sequence as set forth in SEQ ID NO: 125;     -   fff) a VH sequence as set forth in SEQ ID NO: 127;     -   ggg) a VH sequence as set forth in SEQ ID NO: 129;     -   hhh) a VH sequence as set forth in SEQ ID NO: 131;     -   iii) a VH sequence as set forth in SEQ ID NO: 133;     -   jjj) a VH sequence as set forth in SEQ ID NO: 135;     -   kkk) a VH sequence as set forth in SEQ ID NO: 137;     -   lll) a VH sequence as set forth in SEQ ID NO: 139;     -   mmm) a VH sequence as set forth in SEQ ID NO: 141;     -   nnn) a VH sequence as set forth in SEQ ID NO: 143;     -   ooo) a VH sequence as set forth in SEQ ID NO: 145;     -   ppp) a VH sequence as set forth in SEQ ID NO: 147;     -   qqq) a VH sequence as set forth in SEQ ID NO: 149;     -   rrr) a VH sequence as set forth in SEQ ID NO: 151;     -   sss) a VH sequence as set forth in SEQ ID NO: 153;     -   ttt) a VH sequence as set forth in SEQ ID NO: 155;     -   uuu) a VH sequence as set forth in SEQ ID NO: 157;     -   vvv) a VH sequence as set forth in SEQ ID NO: 159;     -   www) a VH sequence as set forth in SEQ ID NO: 161;     -   xxx) a VH sequence as set forth in SEQ ID NO: 163;     -   yyy) a VH sequence as set forth in SEQ ID NO: 165;     -   zzz) a VH sequence as set forth in SEQ ID NO: 167;     -   aaaa) a VH sequence as set forth in SEQ ID NO: 169;     -   bbbb) a VH sequence as set forth in SEQ ID NO: 171;     -   cccc) a VH sequence as set forth in SEQ ID NO: 173;     -   dddd) a VH sequence as set forth in SEQ ID NO: 175;     -   eeee) a VH sequence as set forth in SEQ ID NO: 177;     -   ffff) a VH sequence as set forth in SEQ ID NO: 179;     -   gggg) a VH sequence as set forth in SEQ ID NO: 181;     -   hhhh) a VH sequence as set forth in SEQ ID NO: 183;     -   iiii) a VH sequence as set forth in SEQ ID NO: 185;     -   jjjj) a VH sequence as set forth in SEQ ID NO: 187;     -   kkkk) a VH sequence as set forth in SEQ ID NO: 189;     -   llll) a VH sequence as set forth in SEQ ID NO: 191;     -   mmmm) a VH sequence as set forth in SEQ ID NO: 193;     -   nnnn) a VH sequence as set forth in SEQ ID NO: 195;     -   oooo) a VH sequence as set forth in SEQ ID NO: 197;     -   pppp) a VH sequence as set forth in SEQ ID NO: 199;     -   qqqq) a VH sequence as set forth in SEQ ID NO: 201;     -   rrrr) a VH sequence as set forth in SEQ ID NO: 203;     -   ssss) a VH sequence as set forth in SEQ ID NO: 205;     -   tttt) a VH sequence as set forth in SEQ ID NO: 207;     -   uuuu) a VH sequence as set forth in SEQ ID NO: 209;     -   vvvv) a VH sequence as set forth in SEQ ID NO: 211;     -   wwww) a VH sequence as set forth in SEQ ID NO: 213;     -   xxxx) a VH sequence as set forth in SEQ ID NO: 215;     -   yyyy) a VH sequence as set forth in SEQ ID NO: 217;     -   zzzz) a VH sequence as set forth in SEQ ID NO: 219;     -   aaaaa) a VH sequence as set forth in SEQ ID NO: 221;     -   bbbbb) a VH sequence as set forth in SEQ ID NO: 223;     -   ccccc) a VH sequence as set forth in SEQ ID NO: 225;     -   ddddd) a VH sequence as set forth in SEQ ID NO: 227;     -   eeeee) a VH sequence as set forth in SEQ ID NO: 229;     -   fffff) a VH sequence as set forth in SEQ ID NO: 221;     -   ggggg) a VH sequence as set forth in SEQ ID NO: 223;     -   hhhhh) a VH sequence as set forth in SEQ ID NO: 225;     -   iiiii) a VH sequence as set forth in SEQ ID NO: 227;     -   jjjjj) a VH sequence as set forth in SEQ ID NO: 229;     -   kkkkk) a VH sequence as set forth in SEQ ID NO: 231;     -   lllll) a VH sequence as set forth in SEQ ID NO: 233;     -   mmmmm) a VH sequence as set forth in SEQ ID NO: 235;     -   nnnnn) a VH sequence as set forth in SEQ ID NO: 237;     -   ooooo) a VH sequence as set forth in SEQ ID NO: 239;     -   ppppp) a VH sequence as set forth in SEQ ID NO: 241;     -   qqqqq) a VH sequence as set forth in SEQ ID NO: 243;     -   rrrrr) a VH sequence as set forth in SEQ ID NO: 245;     -   sssss) a VH sequence as set forth in SEQ ID NO: 247;     -   ttttt) a VH sequence as set forth in SEQ ID NO: 249;     -   uuuuu) a VH sequence as set forth in SEQ ID NO: 251;     -   vvvvv) a VH sequence as set forth in SEQ ID NO: 253;     -   wwwww) a VH sequence as set forth in SEQ ID NO: 255;     -   xxxxx) a VH sequence as set forth in SEQ ID NO: 257;     -   yyyyy) a VH sequence as set forth in SEQ ID NO: 259;     -   zzzzz) a VH sequence as set forth in SEQ ID NO: 261;     -   aaaaaa) a VH sequence as set forth in SEQ ID NO: 263;     -   bbbbbb) a VH sequence as set forth in SEQ ID NO: 265;     -   cccccc) a VH sequence as set forth in SEQ ID NO: 267;     -   dddddd) a VH sequence as set forth in SEQ ID NO: 269;     -   eeeeee) a VH sequence as set forth in SEQ ID NO: 271;     -   ffffff) a VH sequence as set forth in SEQ ID NO: 273;     -   gggggg) a VH sequence as set forth in SEQ ID NO: 275;     -   hhhhhh) a VH sequence as set forth in SEQ ID NO: 277;     -   iiiiii) a VH sequence as set forth in SEQ ID NO: 279;     -   jjjjjj) a VH sequence as set forth in SEQ ID NO: 281;     -   kkkkkk) a VH sequence as set forth in SEQ ID NO: 283;     -   llllll) a VH sequence as set forth in SEQ ID NO: 285;     -   mmmmmm) a VH sequence as set forth in SEQ ID NO: 287;     -   nnnnnn) a VH sequence as set forth in SEQ ID NO: 289;     -   oooooo) a VH sequence as set forth in SEQ ID NO: 291;     -   pppppp) a VH sequence as set forth in SEQ ID NO: 293;     -   qqqqqq) a VH sequence as set forth in SEQ ID NO: 295;     -   rrrrrr) a VH sequence as set forth in SEQ ID NO: 297;     -   ssssss) a VH sequence as set forth in SEQ ID NO: 299 and     -   tttttt) a VH sequence as set forth in SEQ ID NO: 301.

In one embodiment, the present invention relates to a humanized or chimeric antibody binding to human CD3, wherein said antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises one of the VH sequences selected from the group consisting of;

-   -   a) a VH sequence as set forth in SEQ ID NO: 55[T31M],     -   b) a VH sequence as set forth in SEQ ID NO: 59 [T31P],     -   c) a VH sequence as set forth in SEQ ID NO: 107 [N57E]     -   d) a VH sequence as set forth in SEQ ID NO: 177 [H101G],     -   e) a VH sequence as set forth in SEQ ID NO: 185 [H101N],     -   f) a VH sequence as set forth in SEQ ID NO: 221 [G105P],     -   g) a VH sequence as set forth in SEQ ID NO: 237 [S110A],     -   h) a VH sequence as set forth in SEQ ID NO: 245 [S110G],     -   i) a VH sequence as set forth in SEQ ID NO: 285 [Y114M],     -   j) a VH sequence as set forth in SEQ ID NO: 293 [Y114R], and     -   k) a VH sequence as set forth in SEQ ID NO: 299 [Y114V].

In one embodiment of the invention the humanized or chimeric antibody comprises a binding region, wherein said binding region comprises a variable light chain (VL) region, wherein said VL region comprises the CDR1, CDR2, and CDR3 having the CDR sequences selected from the group consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 7;     -   b) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 302,         GTN, 7;     -   c) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 304,         GTN, 7;     -   d) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 306,         GTN, 7;     -   e) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 308,         GTN, 7;     -   f) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 310,         GTN, 7;     -   g) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 312,         GTN, 7;     -   h) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 314,         GTN, 7;     -   i) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 316,         GTN, 7;     -   j) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 318,         GTN, 7;     -   k) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 320,         GTN, 7;     -   l) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 322,         GTN, 7;     -   m) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 324,         GTN, 7;     -   n) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 326,         GTN, 7;     -   o) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 328,         GTN, 7;     -   p) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 330,         GTN, 7;     -   q) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 332;     -   r) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 334;     -   s) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 336;     -   t) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 338;     -   u) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 340;     -   v) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 342;     -   w) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 344;     -   x) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 346;     -   y) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 348;     -   z) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 350;     -   aa) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 352;     -   bb) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 354;     -   cc) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 356;     -   dd) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 358;     -   ee) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 360;     -   ff) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 362;     -   gg) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 364;     -   hh) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 366;     -   ii) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 368;     -   jj) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 370;     -   kk) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 372;     -   ll) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 374;     -   mm) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 376;     -   nn) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 378;     -   oo) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 380;     -   pp) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 382;     -   qq) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 384;     -   rr) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 386;     -   ss) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 388;     -   tt) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 390;     -   uu) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 392 and     -   vv) CDR1, CDR2 and CDR3 sequences as set forth in SEQ ID NO: 6,         GTN, 394.

In another embodiment of the invention the humanized or chimeric antibody comprises a binding region comprising a variable light chain (VL) region, wherein said VL region comprises one of the VL sequences selected from the group consisting of;

-   -   a) a VL sequence as set forth in SEQ ID NO:8; and     -   b) a VL sequence as set forth in SEQ ID NO:10;

The term “antibody” as used herein is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an effector activity). The binding region (or binding domain which may also be used herein, both terms having the same meaning) which interacts with an antigen, comprises variable regions of both the heavy and light chains of the immunoglobulin molecule. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system such as C1q, the first component in the classical pathway of complement activation. As indicated above, the term antibody as used herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that retain the ability to specifically interact, such as bind, to the antigen. It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antibody” include (i) a Fab′ or Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, or a monovalent antibody as described in WO2007059782 (Genmab A/S); (ii) F(ab′)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting essentially of the VH and CH1 domains; and (iv) a Fv fragment consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they may be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see for instance Bird et al., Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless otherwise noted or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. These and other useful antibody fragments in the context of the present invention are discussed further herein. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

The term “immunoglobulin heavy chain”, “heavy chain of an immunoglobulin” or “heavy chain” as used herein is intended to refer to one of the chains of an immunoglobulin. A heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) which defines the isotype of the immunoglobulin. The heavy chain constant region typically is comprised of three domains, CH1, CH2, and CH3. The heavy chain constant region may further comprise a hinge region. The term “immunoglobulin” as used herein is intended to refer to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) chains and one pair of heavy (H) chains, all four potentially inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized (see for instance [14]). Within the structure of the immunoglobulin (e.g. IgG), the two heavy chains are inter-connected via disulfide bonds in the so-called “hinge region”. Equally to the heavy chains each light chain is typically comprised of several regions; a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region typically is comprised of one domain, CL. Furthermore, the VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see [15]). CDR sequences may be determined by use of the method provided by IMGT [16]-[17].

The term “isotype” as used herein, refers to the immunoglobulin (sub)class (for instance IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) or any allotype thereof, such as IgG1m(za) and IgG1m(f) [SEQ ID NO:407]) that is encoded by heavy chain constant region genes. Thus, in one embodiment, the antibody comprises a heavy chain of an immunoglobulin of the IgG1 class or any allotype thereof. Further, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

The term “chimeric antibody” as used herein, refers to an antibody wherein the variable region is derived from a non-human species (e.g. derived from rodents) and the constant region is derived from a different species, such as human. Chimeric antibodies may be generated by antibody engineering. “Antibody engineering” is a generic term used for different kinds of modifications of antibodies, and which is a well-known process for the skilled person. In particular, a chimeric antibody may be generated by using standard DNA techniques as described in [18]. Thus, the chimeric antibody may be a genetically engineered recombinant antibody. Some chimeric antibodies may be both genetically or an enzymatically engineered. It is within the knowledge of the skilled person to generate a chimeric antibody, and thus, generation of the chimeric antibody according to the present invention may be performed by other methods than described herein. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce antibody immunogenicity. They may typically contain non-human (e.g. murine) variable regions, which are specific for the antigen of interest, and human constant antibody heavy and light chain domains. The terms “variable region” or “variable domains” as used in the context of chimeric antibodies, refers to a region which comprises the CDRs and framework regions of both the heavy and light chains of the immunoglobulin.

The term “humanized antibody” as used herein, refers to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see [19]-[20]). In order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modeling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties.

The humanized or chimeric antibody according to any aspect or embodiment of the present invention may be termed “humanized or chimeric CD3 antibody”, “humanized or chimeric antibody of the invention”, “CD3 antibody”, or “CD3 antibody of the invention”, which all have the same meaning and purpose unless otherwise contradicted by context.

The amino acid sequence of an antibody of non-human origin is distinct from antibodies of human origin, and therefore a non-human antibody is potentially immunogenic when administered to human patients. However, despite the non-human origin of the antibody, its CDR segments are responsible for the ability of the antibody to bind to its target antigen and humanization aims to maintain the specificity and binding affinity of the antibody. Thus, humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity in man while such humanized antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin.

The term “binding region” as used herein, refers to a region of an antibody which is capable of binding to any molecule, such as a polypeptide, e.g. present on a cell, bacterium, or virion.

The term “binding” as used herein, refers to the binding of an antibody to a predetermined antigen or target to which binding typically is with an affinity corresponding to a K_(D) of about 10⁻⁶ M or less, e.g. 10⁻⁷ M or less, such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less, about 10⁻¹⁰ M or less, or about 10⁻¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a K_(D) that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1,000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The degree with which the affinity is lower is dependent on the K_(D) of the antibody, so that when the K_(D) of the antibody is very low (that is, the antibody is highly specific), then the degree with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold. The term “K_(D)” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

The term “human CD3” as used herein, refers to the human Cluster of Differentiation 3 protein which is part of the T cell co-receptor protein complex and is composed of four distinct chains. CD3 is also found in other species, and thus, the term “CD3” may be used herein and is not limited to human CD3 unless contradicted by context. In mammals, the complex contains a CD3γ (gamma) chain (human CD3γ chain Swissprot P09693, or cynomolgus monkey CD3γ Swissprot Q95LI7), a CD3δ (delta) chain (human CD3δ Swissprot P04234, or cynomolgus monkey CD3δ Swissprot Q95LI8), two CD3ε (epsilon) chains (human CD3ε Swissprot P07766; or cynomolgus CD3ε Swissprot Q95LI5), rhesus CD3ε (Swissprot G7NCB9), and a CD3-chain (zeta) chain (human CD3ζ Swissprot P20963, cynomolgus monkey CD3ζ Swissprot Q09TKO). These chains associate with a molecule known as the T cell receptor (TCR) and generate an activation signal in T lymphocytes. The TCR and CD3 molecules together comprise the TCR complex.

It is within the knowledge of the skilled person that amino acid sequences referred to as Swissprot numbers include a signal peptide which is removed after translation of the protein. Thus, proteins, such as CD3, present on cell surfaces do not include the signal peptide. In particular, the amino acid sequences listed in Table 1 do not contain such signal peptide. Such proteins as listed in Table 1 may be termed “mature proteins”. Thus, SEQ ID NO:398 shows the amino acid sequence of mature human CD3δ (delta), SEQ ID NO:399 shows the amino acid sequence of mature human CD3ε (epsilon), SEQ ID NO:403 shows the amino acid sequence of mature cynomolgus CD3ε, and SEQ ID NO:404 shows the amino acid sequence of mature rhesus CD3ε. Thus, the term “mature” as used herein, refers to a protein which does not comprise any signal or leader sequence.

It is well-known that signal peptide sequence homology, length, and the cleavage site position, varies significantly between different proteins. Signal peptides may be determined by different methods, e.g. SEQ ID NO:399 of the present invention has been determined according to the SignalP application (available on www.cbs.dtu.dk/services/SignalP/).

In a particular embodiment, the humanized or chimeric antibody of the present invention binds the epsilon chain of CD3, such as the epsilon chain of human CD3 (SEQ ID NO:399). In yet another particular embodiment, the humanized or chimeric antibody binds an epitope within amino acids 1-27 of the N-terminal part of human CD3ε (epsilon) (SEQ ID NO:402). In such a particular embodiment, the antibody may even further cross-react with other non-human primate species, such as cynomolgus monkeys (cynomolgus CD3 epsilon SEQ ID NO:403) and/or rhesus monkeys (rhesus CD3 epsilon SEQ ID NO:404).

An antibody according to the present invention comprising the CDR sequences as defined herein, further comprising framework regions may differ in sequence outside the CDR sequences but still retains the full binding ability as compared to the original antibody. Thus, the present invention also relates to antibodies comprising an amino acid sequence of the variable region having a certain sequence identity to any sequence herein described.

The term “sequence identity” as used in the context of the present invention, refers to the percent identity between two sequences as a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g. be determined using the algorithm of E. Meyers and W. Miller [21]. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch algorithm [22]. Multiple alignments are preferably performed using the Clustal W algorithm [23] (as used e.g., in Vector NTI Advance® software version 11.5; Invitrogen Inc.).

Thus, in one embodiment of the present invention, the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the three CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2,         3 [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114N1];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R];     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V]; and     -   l) CDR1, CDR2 and CDR3 sequences having at least 90% or at least         95% amino acid sequence identity, in total across the three CDR         sequences, to any one of the three CDR sequences as set forth         in a) to k), provided that the CDR1, CDR2 and CDR3 sequences do         not have the sequences as set forth in SEQ ID NO: 1, 2, 3.

The VH region, as illustrate in the sequence table 1 of the present document, consists of 125 amino acid sequence. Thus a second VH sequence consisting of 125 amino acids whereof 124 amino acid positions are identical with the one of the first VH sequences listed above has 99.2% sequence identity with said first VH sequence. A second sequence consisting of 125 amino acids whereof 120 amino acid positons are identical with one of the first VH sequences listed above have 96% sequence identity with said first VH sequence. A second sequence consisting of 125 amino acids whereof 115 amino acid positions are identical with one of the first VH sequences listed above have 92% sequence identity with said first VH sequence.

In a particular embodiment thereof, the VH region has at least 96% amino acid sequence identity to at least one of the VH sequences as specified in said group.

In one embodiment of the invention the mutations are located in the frame work regions of the VH region. Hence, in some embodiments the three CDR sequences of the VH region are 100% identical to the antibodies of the present invention, but amino acid variation may occurred in the frame work region of the VH region. Such amino acid variation in the frame work region may preferably not change the binding affinity of the antibody to CD3 compare to the antibody when the CDRs are comprised in the reference frame of SEQ ID NO: 407.

The mutations in the VH sequence causing variations in the sequence identity may preferably be conservative, physical or functional amino acids. Substituting amino acids with similar amino acids may increase the likelihood of keeping the functionality of the parent antibody.

In one embodiment of the invention the antibody is a humanized antibody.

In one embodiment of the invention the antibody is a full-length antibody.

The humanized antibody according to the present invention may be generated by comparison of the heavy and light chain variable region amino acid sequences against a database of human germline variable region sequences in order to identify the heavy and light chain human sequence with the appropriate degree of homology for use as human variable framework regions. A series of humanized heavy and light chain variable regions may be designed by grafting, e.g. the murine, CDRs onto the framework regions (identified as described above) and, if necessary, by back-mutation (mutation of one or more of the human amino acid residues in the framework regions back to the non-human amino acid at the specific position(s)) to the specific murine sequence of residues identified which may be critical to the restoration of the antibody binding efficiency. Variant sequences with the lowest incidence of potential T cell epitopes may then be selected as determined by application of in silico technologies; iTope™ and TCED™ ([24], [25], and [26]).

-   -   Furthermore, the humanized antibodies according to the present         invention may also be “deimmunized”. Deimmmunization may be         desired, as within a protein sequence, such as a humanized         antibody according to the present invention, the presence of         human T cell epitopes may increase the immunogenicity risk         profile as they have the potential to activate helper T cells.         Such activation of helper T cells may be avoided by         deimmunization. Deimmunization may be performed by introducing a         mutation in the amino acid sequence of the humanized antibody in         order to remove the T cell epitopes without significantly         reducing the binding affinity of the antibody.     -   Thus, in one embodiment of the present invention, the humanized         antibody may be produced by a method comprising the steps of (i)         comparing the non-human full variable heavy chain sequence         and/or the full variable light chain sequence to a database of         human germline sequences, (ii) selecting the human germline         sequence having the highest homology to the non-human sequence         to obtain a humanized sequence, (iii) optimizing the humanized         sequence by back-mutation(s) if required, and (iv) expressing         the sequence in a suitable expression system.     -   Thus, a full-length antibody according to the present invention         may be produced by a method comprising the steps of (i)         comparing the non-human variable heavy chain sequence and the         variable light chain sequences to a database of human germline         sequences, (ii) selecting the human germline sequence having the         highest homology to the non-human sequence, (iii) grafting of         the non-human CDRs in to the selected human germ-line to obtain         a humanized sequence, (iv) optimizing the humanized sequences by         back-mutation(s) if required, (v) identifying constant heavy and         light chain sequences, and (vi) expressing the complete heavy         chain sequences and complete light chain sequences in suitable         expression systems. A full-length antibody according to the         present invention may, thus, be produced as described in         Example 1. It is within the knowledge of the skilled person to         produce a full-length antibody when starting out from either CDR         sequences or full variable region sequences. Thus, the skilled         person would know how to generate a full-length antibody         according to the present invention.     -   The term “complete heavy chain sequences” as used herein, refers         to a sequence consisting of variable heavy chain and constant         heavy chain sequences.     -   The term “complete light chain sequences” as used herein, refers         to a sequence consisting of variable light chain and constant         light chain sequences.     -   Back-mutation(s) may be introduced by standard DNA mutagenesis.         Such standard techniques for DNA mutagenesis are described in         [18]. Alternatively, use of commercially available kits such as         Quickchange™ Site-Directed Mutagenesis Kit (Stratagene), or the         desired back-mutations may be introduced by de novo DNA         synthesis.     -   Thus, in one embodiment, the antibody is a humanized antibody.     -   Chimeric antibodies may be generated by substituting all         constant region sequences of a non-human (such as murine)         antibody with constant region sequences of human origin. Thus,         fully non-human variable region sequences are maintained in the         chimeric antibody. Thus, a chimeric antibody according to the         present invention may be produced by a method comprising the         step of expressing the non-human variable heavy chain (SEQ ID         NO:405), non-human variable light chain sequences (SEQ ID         NO:406), human constant heavy chain and human constant light         chain sequences in suitable expression systems, and thereby         generating a full-length chimeric antibody. Alternative methods         may be used. Such methods of producing a chimeric antibody is         within the knowledge of the skilled person, and thus, the         skilled person would know how to produce a chimeric antibody         according to the present invention. Thus to make a chimeric         antibody according to the present invention one would introduce         the mutations according to the invention in the non-human (such         as murine) VH or VL sequence.     -   Thus, in one embodiment, the antibody is a chimeric antibody.     -   In one embodiment, the antibody is a full-length antibody. The         term “full-length antibody” as used herein, refers to an         antibody (e.g., a parent or variant antibody) which contains all         heavy and light chain constant and variable domains correspond         to those that are normally found in a wild-type antibody of that         isotype.     -   In one embodiment, the antibody comprises an Fc region         comprising a first and a second immunoglobulin heavy chain.     -   The term “Fc region” as used herein, refers to a region         comprising, in the direction from the N- to C-terminal, at least         a hinge region, a CH2 region and a CH3 region. An Fc region may         further comprise a CH1 region at the N-terminal end of the hinge         region.     -   The term “hinge region” as used herein refers to the hinge         region of an immunoglobulin heavy chain. Thus, for example the         hinge region of a human IgG1 antibody corresponds to amino acids         216-230 according to the Eu numbering as set forth in Kabat.     -   Unless otherwise stated or contradicted by context, the amino         acids of the constant region sequences are herein numbered         according to the Eu-index of numbering (described in [27]) and         may be termed “according to the Eu numbering as set forth in         Kabat”, “Eu numbering according to Kabat”, or “according to the         Eu numbering system”.     -   The term “CH1 region” or “CH1 domain” as used herein, refers to         the CH1 region of an immunoglobulin heavy chain. Thus, for         example the CH1 region of a human IgG1 antibody corresponds to         amino acids 118-215 according to the Eu numbering system.         However, the CH1 region may also be any of the other subtypes as         described herein.     -   The term “CH2 region” or “CH2 domain” as used herein, refers to         the CH2 region of an immunoglobulin heavy chain. Thus, for         example the CH2 region of a human IgG1 antibody corresponds to         amino acids 231-340 according to the Eu numbering system.         However, the CH2 region may also be any of the other subtypes as         described herein.     -   The term “CH3 region” or “CH3 domain” as used herein, refers to         the CH3 region of an immunoglobulin heavy chain. Thus, for         example the CH3 region of a human IgG1 antibody corresponds to         amino acids 341-447 according to the Eu numbering system.         However, the CH3 region may also be any of the other subtypes as         described herein.     -   In one embodiment, the isotype of the immunoglobulin heavy chain         is selected from the group consisting of IgG1, IgG2, IgG3, and         IgG4. The immunoglobulin heavy chain may be any allotype within         each of the immunoglobulin classes, such as IgG1m(f) (SEQ ID         NO:407). Thus, in one particular embodiment, the isotype of the         immunoglobulin heavy chains is an IgG1, or any allotype thereof,         such as IgG1m(f) (SEQ ID NO:407).     -   When targeting the antigen CD3 which is part of the T cell         Receptor (TCR), the T cell specific mechanisms of cell killing         is desirable. Other effector functions, e.g. complement         activation, may not be wanted, and therefore, reduction of         effector functions is desirable. C1q binding is the first step         in the complement cascade, and therefore serves as an indicator         for complement-dependent cytotoxicity (CDC) capacity of         antibodies. If binding of C1q to the antibody can be avoided,         activation of the complement cascade can be avoided as well.     -   Thus, in one embodiment, the antibody comprises an Fc region         which has been modified so that binding of C1q to said antibody         is reduced compared to a wild-type antibody by at least 70%, at         least 80%, at least 90%, at least 95%, at least 97%, at least         99%, at least 99.9% or 100%, wherein C1q binding is determined         by ELISA. In a preferred embodiment the antibody comprises an Fc         region, which has been modified so that the binding of C1q to         said antibody is reduced compared to a wild-type antibody by at         least 99% to a 100%, wherein C1q binding is determined by ELISA.     -   The term “modified” as used herein, refers to the amino acid         sequence of an Fc region which is not identical to the amino         acid sequence of a wild-type Fc region. I.e. amino acid residues         in specific positions of the wild-type Fc region have been         substituted, deleted or inserted in order to alter, for example,         the binding site for C1q, binding site for other effector         molecules or binding to Fc Receptors (FcRs). Such         modification(s) of the amino acid sequence may be prepared by         substituting one or more amino acids with a conservative amino         acid or may be prepared by substituting one or more amino acids         with an alternative amino acid which is physically and/or         functionally similar to the amino acid present in the wild-type.         Substitutions may also be prepared by substituting with a         non-conservative amino acid.     -   In the context of the present invention, amino acids may be         described as conservative or non-conservative amino acids, and         may therefore be classified accordingly. Amino acid residues may         also be divided into classes defined by alternative physical and         functional properties. Thus, classes of amino acids may be         reflected in one or both of the following tables:

Amino acid residue of conservative class Acidic Residues D and E Basic Residues K, R, and H Hydrophilic Uncharged Residues S, T, N, and Q Aliphatic Uncharged Residues G, A, V, L, and I Non-polar Uncharged Residues C, M,and P Aromatic Residues F, Y, and W

Alternative Physical and Functional Classifications of Amino Acid Residues Alcohol group- S and T containing residues Aliphatic residues I, L, V, and M Cycloalkenyl- F, H, W, and Y associated residues Hydrophobic A, C, F, G, H, I, L, M, R, T, residues V, W, and Y Negatively charged D and E residues Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged H, K, and R residues Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved A, C, D, E, G, H, K, N, Q, R, in turn formation S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and R

-   -   In the context of the present invention, a substitution in an         antibody, such as a humanized or chimeric antibody, is indicated         as:     -   Original amino acid-position-substituted amino acid;     -   Referring to the well-recognized nomenclature for amino acids,         the three letter code, or one letter code, is used, including         the codes Xaa and X to indicate any amino acid residue.         Accordingly, the notation “L234F” or “Leu234Phe” means, that the         antibody comprises a substitution of Leucine with Phenylalanine         in amino acid position 234.     -   Substitution of an amino acid at a given position to any other         amino acid is referred to as:     -   Original amino acid-position; or e.g. “L234”.     -   For a modification where the original amino acid(s) and/or         substituted amino acid(s) may comprise more than one, but not         all amino acid(s), the more than one amino acid may be separated         by “,” or “/”. E.g. the substitution of Leucine for         Phenylalanine, Arginine, Lysine or Tryptophan in position 234         is:     -   “Leu234Phe,Arg,Lys,Trp” or “Leu234Phe/Arg/Lys/Trp” or         “L234F,R,K,W” or “L234F/R/K/W” or “L234 to F, R, K or W”     -   Such designation may be used interchangeably in the context of         the invention but have the same meaning and purpose.     -   Furthermore, the term “a substitution” embraces a substitution         into any one of the other nineteen natural amino acids, or into         other amino acids, such as non-natural amino acids. For example,         a substitution of amino acid L in position 234 includes each of         the following substitutions: 234A, 234C, 234D, 234E, 234F, 234G,         234H, 234I, 234K, 234M, 234N, 234Q, 234R, 234S, 234T, 234V,         234W, 234P, and 234Y. This is, by the way, equivalent to the         designation 234X, wherein the X designates any amino acid other         than the original amino acid. These substitutions can also be         designated L234A, L234C, etc., or L234A,C, etc., or L234A/C/etc.         The same applies by analogy to each and every position mentioned         herein, to specifically include herein any one of such         substitutions.     -   The antibody according to the invention may also comprise a         deletion of an amino acid residue. Such deletion may be denoted         “del”, and includes, e.g., writing as L234del. Thus, in such         embodiments, the Leucine in position 234 has been deleted from         the amino acid sequence.     -   The terms “amino acid” and “amino acid residue” may herein be         used interchangeably.

In one embodiment of the invention, the antibody comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 regions having the three CDR sequences selected from one of the groups consisting of;

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2,         3 [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];

e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 184 [H101N];

f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 220 [G105P];

-   -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114M];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R];     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V], and     -   l) CDR1, CDR2 and CDR3 sequences as specified in a) to k) having         at most 5 further mutations or substitutions, at most 4 further         mutations or substitutions, at most 3 further mutations or         substitutions, at most 2 further mutations or substitutions, or         at most 1 further mutation or substitution, in total across the         three CDR sequences, and which mutations or substitutions         preferably do not modify the binding affinity to human CD3.

In one embodiments of the invention the further mutations or substitutions are conservative, physical or functional amino acids.

In some embodiments binding to CD3 may be binding to full length CD3 such as CD3 present on a T cell. In other embodiments binding to CD3 may be binding to a CD3 peptide e.g. as set forth in SEQ ID NO: 402. Binding to the CD3 peptide and whether or not any further mutations may modify binding to CD3 can be determined by Bio-Layer Inerferometry as disclosed in Example 7.

In one embodiment, the antibody comprises an Fc region comprising a first and a second immunoglobulin heavy chain.

-   -   The term “C1q binding” as used herein, refers to the binding of         C1q to an antibody, when said antibody is bound to its antigen.         The term “bound to its antigen” as used herein, refers to         binding of an antibody to its antigen both in vivo and in vitro.     -   The term “reduced” as used herein when referring to C1q binding,         refers to the ability of the antibody according to the invention         to reduce, minimize or even completely inhibit the binding of         C1q to the antibody when compared to the C1q binding to a         wild-type antibody.     -   The term “reduced” or “reducing” as used herein or any variation         thereof when used in relation to binding affinity of an antibody         binding to human CD3, refers to a binding affinity that is lower         when compared to a reference binding affinity. In this context,         the reference binding affinity may be the binding affinity of         reference antibody specified by the VH sequence SEQ ID NO:4 and         the VL sequence SEQ ID NO:8 when binding to the CD3 peptide as         SEQ ID NO: 402 and determined by Bio-Layer Interferomerty as         described in example 7.     -   The term “binding affinity” as used herein refers to the binding         of an antibody to a predetermined antigen or target to which         binding typically is with an affinity corresponding to a K_(D).         The term “K_(D)” (M), as used herein, refers to the dissociation         equilibrium constant of a particular antibody-antigen         interaction.     -   The term “wild-type antibody” as used herein, in relation to use         in comparison assays of an antibody according to the present         invention, refers to an antibody which is identical to the         antibody to be tested except for not being inert. In this         context, the term “inert” refers to a modified Fc region having         reduced or no binding of C1q, i.e. where C1q binding is         determined by ELISA; reduced or no Fc-mediated T cell         proliferation as determined in a PBMC based functional assay,         i.e. T cell proliferation is measured in a peripheral blood         mononuclear cell (PBMC)-based functional assay; and/or reduced         or no Fc-mediated CD69 expression as determined in a PBMC-based         functional assay. Thus, the wild-type antibody comprises the         naturally occurring amino acids in the immunoglobulin heavy         chains, i.e. an antibody which does not comprise any amino acid         modifications which may alter or reduce the ability of the         antibody to interact with e.g. C1q, Fc Receptors or the like.         Thus, such a wild-type antibody will remain an activating         antibody which is able to bind e.g. C1q. A wild-type antibody         and an antibody of the present invention may comprise other         amino acid modifications than those affecting the antibody's         ability of inducing effector functions, in order to make the         antibody a bispecific antibody or the like.     -   The term “ELISA” as used herein refers to enzyme-linked         immunosorbent assay which is a test that uses antibodies and         color change to identify a substance. A first specific antibody         is attached to the plate surface. Thereby the protein from a         sample is added wherein binding to said first specific antibody         is tested. A second antibody binding the antibody from the         sample is added. The second antibody is linked to an enzyme,         and, in the final step, a substance containing the enzyme's         substrate is added. The subsequent reaction produces a         detectable signal, most commonly a color change in the         substrate. The concept of the ELISA method is well-known within         the art and various ways of performing an ELISA are contemplated         to be part of a method to evaluate the antibody according to the         invention     -   Specifically, the ability of an antibody according to the         present invention to bind C1q may be determined by ELISA         comprising the steps of (i) coating said antibody on a 96-well         plate, (ii) adding 3% serum, (iii) adding an anti-human C1q         antibody, (iv) developing the plate, and (v) measuring 0D405 nm.         Thus, in one embodiment, the antibody comprises an Fc region         which has been modified so that binding of C1q to said antibody         is reduced compared to a wild-type antibody by at least 70%, at         least 80%, at least 90%, at least 95%, at least 97%, or 100%,         wherein C1q binding is determined by ELISA comprising the steps         of (i) coating said antibodies on a 96-well plate, (ii) adding         3% serum, (iii) adding an anti-human C1q, (iv) developing the         plate, and (v) measuring 0D405 nm.     -   The terms “Fc Receptor” or “FcR” as used herein, refers to a         protein found on the surface of certain cells. FcRs bind to the         Fc region of antibodies. There are several different types of         FcRs which are classified based on the type of antibody they         recognize. E.g. Fcγ (gamma) Receptors bind to antibodies of the         IgG class.     -   The terms “Fcγ Receptor”, “Fc gamma Receptor” or “FcγR” as used         herein, refers to a group of Fc Receptors belonging to the         immunoglobulin superfamily and is the most important Fc         receptors for inducing phagocytosis of opsonized (coated)         microbes. This family includes several members, FcγRI (CD64),         FcγRIIa (CD32a), FcγRIIb (CD32b), FcγRIIIa (CD16a), FcγRIIIb         (CD16b), which differ in their antibody affinities due to their         different molecular structure.     -   Fc-mediated effector functions form part of the biological         activity of human immunoglobulin G (IgG) molecules. Examples of         such effector functions include e.g. antibody-dependent         cell-mediated cytotoxicity (ADCC) and complement-dependent         cytotoxicity (CDC) which are triggered by the binding of various         effector molecules to the Fc region. In the context of the         present invention, “Fc binding”, “Fc Receptor binding”, “FcR         binding”, and “binding of an antibody Fc region to FcR” refers         to the binding of the Fc region to an Fc Receptor (FcR) or an         effector molecule. The terms “FcγR binding” and “FcγRI binding”         refer to binding to or with an Fc region to the Fc gamma         Receptor and Fc gamma Receptor I, respectively. When a CD3         antibody binds T cells, the wild-type Fc region of the CD3         antibody binds to FcRs present on other cells, e.g. monocytes,         which leads to non-specific, Fc-mediated activation of the T         cell. Such non-specific, Fc-mediated activation of T cells may         be undesired. T cells may also be activated by targeted, or         target-specific, T cell activation. Such targeted T cell         activation may be highly desirable for the treatment of a range         of indications, such as cancer. The term “targeted T cell         activation” as used herein, refers to directing the T cells to         specific cells, such as tumor cells by use of a bispecific         antibody comprising a first binding region binding a specific         target, such as a tumor target on a tumor cell, and a second         binding region binding a T cell specific target, such as CD3.         Thus, targeting of T cells to specific cells, e.g. tumor cells,         may be facilitated by use of a bispecific antibody, wherein one         of the binding regions binds CD3 present on the T cell and the         other binding region binds a target specific antigen, e.g. on a         tumor cell. Although, non-specific, Fc-mediated T cells         activation may still be possible and therefore such undesired         non-specific, Fc-mediated T cell activation via Fc-mediated         cross-linking should be avoided and may be disabled by making         the Fc region inert for such activity. Thereby, interaction         between said inert Fc region with Fc Receptors present is         prevented.     -   An antibody according to the present invention may comprise         modifications in the Fc region. When an antibody comprises such         modifications it may become an inert, or non-activating,         antibody. The term “inertness”, “inert” or “non-activating” as         used herein, refers to an Fc region which is at least not able         to bind any Fcγ Receptors, induce Fc-mediated cross-linking via         FcRs, or induce FcR-mediated cross-linking of target antigens         via the Fc region, or is not able to bind C1q. The inertness of         an Fc region of a humanized or chimeric CD3 antibody is         advantageously tested using the antibody in a monospecific         format although an inert Fc region so identified can be used in         bispecific or other humanized or chimeric multispecific CD3         antibodies.     -   Several variants can be constructed to make the Fc region of an         antibody inactive for interactions with Fc gamma Receptors and         C1q for therapeutic antibody development. Examples of such         variants are described herein.     -   Thus, in one embodiment, the antibody comprises an Fc region         which has been modified so that said antibody mediates reduced         Fc-mediated T cell proliferation compared to a wild-type         antibody by at least 50%, at least 60%, at least 70%, at least         80%, at least 90%, at least 99% or 100%, wherein said T cell         proliferation is measured in a peripheral blood mononuclear cell         (PBMC)-based functional assay.     -   The term “reduce” when referring to T cell proliferation, refers         to the ability of the antibody according to the invention to         reduce, minimize or even completely inhibit the proliferation of         T cells when compared to the proliferation of T cells bound by a         wild-type antibody. The ability of an antibody to reduce T cell         proliferation may be evaluated by a PBMC-based functional assay.         In one embodiment the assay is performed with human PBMCs. In         another embodiment the assay is performed with cynomolgus PBMCs.         In yet another embodiment, the assay is performed with rhesus         PBMCs. Since the antibodies according to the present invention         are cross-reactive, a PBMC-based assay as herein described may         be performed with any species PBMCs to show reduction of T cell         proliferation as long as the species PBMC used are within the         cross-reactivity spectra of the antibodies, e.g. human,         cynomolgus or rhesus monkeys.     -   The term “peripheral blood mononuclear cell (PBMC)-based         functional assay” as used herein refers to an assay used for         evaluating a functional feature of the antibody of the present         invention, such as the ability of said antibody to affect T cell         proliferation or CD69 expression, wherein the only cells present         are peripheral blood mononuclear cells. Thus, in one embodiment,         T cell proliferation is measured by a method comprising the         steps of incubating PBMCs with antibody in the range of 1-1000         ng/mL at 37° C. in a 5% (vol/vol) CO₂ humidified incubator for         three days, adding a chemical compound, such as BrdU, which is         incorporated into the DNA of proliferating cells, incubating for         five hrs., pelleting cells, drying cells, optionally storing the         cells at 4° C., coating cells to ELISA plates, incubating with         anti-BrdU-peroxidase for 90 min at room temperature, developing         for about 30 min with 1 mg/mL 2,2′-azino-bis         (3-ethylbenzothiazoline-6-sulfonic acid), adding 100 μL 2%         oxalic acid to stop the reaction, and measuring absorbance at         405 nm in a suitable microplate reader.     -   The term “proliferation” as used herein, refers to cell growth         in the context of cell division.     -   The term “BrdU” as used herein, refers to         5-bromo-2′-deoxyuridine, which is a homologue to thymidine. When         BrdU is added to cell culture for a limited period of time (e.g.         4 hours) it will be incorporated into the DNA of proliferating         cells. After fixing the cells, detection of incorporated BrdU         may be performed in an ELISA using anti-BrdU-peroxidase. BrdU         incorporation is therefore a measure for proliferation.     -   In one embodiment, the antibody comprises an Fc region which has         been modified so that said antibody reduces Fc-mediated CD69         expression by at least 50%, at least 60%, at least 70%, at least         80%, at least 90%, at least 99% or 100% when compared to a         wild-type antibody wherein said Fc-mediated CD69 expression is         determined in a PBMC-based functional assay.     -   In particular, the term “reduce” when referring to expression         level of the T cell activation marker CD69, refers to a         reduction in expression level of CD69 when compared to         expression level of CD69 when the T cell is bound by a wild-type         antibody bound to CD3 and interacting with an Fc receptor. An         antibody's ability to reduce expression of CD69 may be evaluated         by a PBMC-based functional. Thus, in one embodiment, expression         of CD69 is measured by a method comprising the steps of         incubating PBMCs with an antibody in the range of 1-1000 ng/mL         at 37° C. in a 5% (vol/vol) CO₂ humidified incubator for 16-24         hrs, washing the cells, staining the cells at 4° C. with a mouse         anti-human CD28-PE and mouse-anti-human CD69-APC antibody, and         determining CD69-expression on CD28 positive cells by flow         cytometry.     -   The term “CD69” as used herein, refers to Cluster of         Differentiation 69 which is a human transmembrane C-Type lectin         protein encoded by the CD69 gene. Activation of T lymphocytes         and natural killer (NK) cells, both in vivo and in vitro,         induces expression of CD69. CD69 function as a signal         transmitting receptor involved in cellular activation events         including proliferation, functions as a signal-transmitting         receptor in lymphocytes, including natural killer cells and         platelets, and the induction of specific genes.     -   The term “peripheral blood mononuclear cell (PBMC)-based         functional assay” as used herein refers to an assay used for         evaluating a functional feature of the antibody of the present         invention, such as the ability of said antibody to affect T cell         proliferation or CD69 expression, wherein the only cells present         are peripheral blood mononuclear cells. A PBMC-based functional         assay comprises the steps of (i) incubating PBMCs with an         antibody at 37° C. in a 5% (vol/vol) CO₂ humidified incubator         for about 16-24 hrs, (ii) washing the cells, (iii) staining the         cells at 4° C. with a mouse anti-human CD28-PE and         mouse-anti-human CD69-APC antibody, and (iv) determining the         CD69 expression on CD28 positive cells by flow cytometry, when         CD69 expression is evaluated.     -   Amino acids in the Fc region that play a dominant role in the         interactions with C1q and the Fc Gamma Receptors may be         modified. Examples of amino acid positions that may be modified         include positions L234, L235 and P331. Combinations thereof,         such as L234F/L235E/P331S, can cause a profound decrease in         binding to human CD64, CD32A, CD16 and C1q.     -   Hence, in one embodiment, the amino acid in at least one         position corresponding to L234, L235 and P331, may be A, A and         S, respectively ([1], [28]). Also, L234F and L235E amino acid         substitutions can result in Fc regions with abrogated         interactions with Fc Gamma Receptors and C1q ([29]-[30]). Hence,         in one embodiment, the amino acids in the positions         corresponding to L234 and L235, may be F and E, respectively. A         D265A amino acid substitution can decrease binding to all Fc         gamma Receptors and prevent ADCC ([31]). Hence, in one         embodiment, the amino acid in the position corresponding to D265         may be A. Binding to C1q can be abrogated by mutating positions         D270, K322, P329, and P331. Mutating these positions to either         D270A or K322A or P329A or P331A can make the antibody deficient         in CDC activity ([32]). Hence, in one embodiment, the amino         acids in at least one position corresponding to D270, K322, P329         and P331, may be A, A, A, and A, respectively.     -   An alternative approach to minimize the interaction of the Fc         region with Fc gamma Receptors and C1q is by removal of the         glycosylation site of an antibody. Mutating position N297 to         e.g. Q, A, and E removes a glycosylation site which is critical         for IgG-Fc gamma Receptor interactions. Hence, in one         embodiment, the amino acid in a position corresponding to N297,         may be G, Q, A or E ([33]). Another alternative approach to         minimize interaction of the Fc region with Fc gamma Receptors         may be obtained by the following mutations; P238A, A327Q, P329A         or E233P/L234V/L235A/G236del ([31]).     -   Alternatively, human IgG2 and IgG4 subclasses are considered         naturally compromised in their interactions with C1q and Fc         gamma Receptors although, interactions with Fcγ Receptors (Fc         gamma Receptors) were reported ([34]-[35]). Mutations abrogating         these residual interactions can be made in both isotypes,         resulting in reduction of unwanted side-effects associated with         FcR binding. For IgG2, these include L234A and G237A, and for         IgG4, L235E. Hence, in one embodiment, the amino acid in a         position corresponding to L234 and G237 in a human IgG2 heavy         chain, may be A and A, respectively. In one embodiment, the         amino acid in a position corresponding to L235 in a human IgG4         heavy chain, may be E.     -   Other approaches to further minimize the interaction with Fc         gamma Receptors and C1q in IgG2 antibodies include those         described in [36] and [37].     -   The hinge region of the antibody can also be of importance with         respect to interactions with Fc gamma Receptors and complement         ([38]-[39]). Accordingly, mutations in or deletion of the hinge         region can influence effector functions of an antibody.     -   The term “cross-linking” as used herein, refers to the indirect         bridging of antibody Fab arm(s) (monovalently or bivalently)         bound to the target antigen by FcR-bearing cell through binding         to the antibody Fc region. Thus, an antibody which binds its         target antigen on target antigen-bearing cells may cross-link         with another cell expressing FcRs.     -   The term “unspecific killing” as used herein, refers to the         killing of cells by the cytotoxic function of T cells or other         effector cells, through tumor target antigen-independent         activation of said cells. Thus, by unspecific killing is meant         that the tumor-target bearing cells may be killed by e.g.         cytotoxic T cells and not by the antibody binding the tumor         target by e.g. induction of CDC.     -   A non-activating Fc region may be obtained by modifying one or         more of at least five specific amino acid positions in the Fc         region.     -   In one embodiment, the antibody comprises an Fc region         comprising a first and a second immunoglobulin heavy chain.     -   Thus, in one embodiment, the antibody comprises a first and a         second immunoglobulin heavy chain, wherein in at least one of         said first and second immunoglobulin heavy chains one or more         amino acids in the positions corresponding to positions L234,         L235, D265, N297, and P331 in a human IgG1 heavy chain, are not         L, L, D, N, and P, respectively.     -   In one embodiment, in both the first and second heavy chains one         or more amino acids in the position corresponding to positions         L234, L235, D265, N297, and P331 in a human IgG1 heavy chain,         are not L, L, D, N, and P, respectively.     -   In another embodiment, in at least one of the first and second         heavy chains one or more amino acids in the positions         corresponding to positions L234, L235 and D265 in a human IgG1         heavy chain, are not L, L and D, respectively, and the amino         acids in the positions corresponding to N297 and P331 in a human         IgG1 heavy chain, are N and P, respectively.     -   The term “amino acid corresponding to positions” as used herein         refers to an amino acid position number in a human IgG1 heavy         chain. Unless otherwise stated or contradicted by context, the         amino acids of the constant region sequences are herein numbered         according to the Eu-index of numbering (described in [27]).         Thus, an amino acid or segment in one sequence that “corresponds         to” an amino acid or segment in another sequence is one that         aligns with the other amino acid or segment using a standard         sequence alignment program such as ALIGN, ClustalW or similar,         typically at default settings and has at least 50%, at least         80%, at least 90%, or at least 95% identity to a human IgG1         heavy chain. It is considered well-known in the art how to align         a sequence or segment in a sequence and thereby determine the         corresponding position in a sequence to an amino acid position         according to the present invention.     -   In the context of the present invention, the amino acid may be         defined as described above.     -   The term “the amino acid is not” or similar wording when         referring to amino acids in a heavy chain is to be understood to         mean that the amino acid is any other amino acid than the         specific amino acid mentioned. For example, the amino acid in         the position corresponding to L234 in a human IgG1 heavy chain         is not L, means that the amino acid may be any of the other         naturally or non-naturally occurring amino acids than L.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acid in the position corresponding to         position D265 in a human IgG1 heavy chain, is not D.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acid in the position corresponding to D265 in a         human IgG1 heavy chain, is not D, and the amino acids in the         positions corresponding to positions N297 and P331 in a human         IgG1 heavy chain, are N and P, respectively.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         position D265 in a human IgG1 heavy chain is hydrophobic or         polar amino acids.     -   The term “hydrophobic” as used herein in relation to an amino         acid residue, refers to an amino acid residue selected from the         group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y.         Thus, in one embodiment, in at least one of said first and         second heavy chains the amino acid in the position corresponding         to position D265 in a human IgG1 heavy chain is selected from         the group of amino acids consisting of; A, C, F, G, H, I, L, M,         R, T, V, W and Y.     -   The term “polar” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in         one embodiment, in at least one of said first and second heavy         chains the amino acid in the position corresponding to position         D265 in a human heavy chain is selected from the group         consisting of; C, E, H, K, N, Q, R, S, and T.     -   In another embodiment, in at least one of said first and second         heavy chains the amino acid in the position corresponding to         position D265 in a human IgG1 heavy chain is an aliphatic         uncharged, aromatic or acidic amino acid.     -   The term “aliphatic uncharged” as used herein in relation to         amino acid residues, refers to any amino acid residue selected         from the group consisting of: A, G, I, L, and V. Thus, in one         embodiment, in at least one of said first and second heavy         chains the amino acid in the position corresponding to position         D265 in a human IgG1 heavy chain is selected from the group         consisting of; A, G, I, L, and V.     -   The term “aromatic” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of: F, T, and W. Thus, in one embodiment, in at         least one of said first and second heavy chains the amino acid         in the position corresponding to position D265 in a human IgG1         heavy chain is selected from the group consisting of; F, T, and         W.     -   The term “acidic” as used herein in relation to amino acid         residues, refers to any amino acid residue chosen from the group         consisting of: D and E. Thus, in one embodiment, in at least one         of said first and second heavy chains the amino acid in the         position corresponding to position D265 in a human IgG1 heavy         chain is selected from the group consisting of; D and E.     -   In a particular embodiment, in at least one of said first and         second heavy chains the amino acid in the position corresponding         to position D265 in a human IgG1 heavy chain is selected from         the group consisting of; A, E, F, G, I, L, T, V, and W.     -   In one embodiment, in both said first and second heavy chains         the amino acid in the position corresponding to position D265 in         a human IgG1 heavy chain, is not D.     -   In one embodiment, in both the first and second heavy chains the         amino acid in the position corresponding to D265 in a human IgG1         heavy chain, is not D, and the amino acids in the positions         corresponding to positions N297 and P331 in a human IgG1 heavy         chain, are N and P, respectively.     -   In one embodiment, in both said first and second heavy chains         the amino acid in the position corresponding to position D265 in         a human IgG1 heavy chain is hydrophobic or polar amino acid.     -   The term “hydrophobic” as used herein in relation to an amino         acid residue, refers to an amino acid residue selected from the         group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y.         Thus, in one embodiment, in both said first and second heavy         chains the amino acid in the position corresponding to position         D265 in a human IgG1 heavy chain is selected from the group of         amino acids consisting of; A, C, F, G, H, I, L, M, R, T, V, W         and Y.     -   The term “polar” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in         one embodiment, in both said first and second heavy chains the         amino acid in the position corresponding to position D265 in a         human heavy chain is selected from the group consisting of; C,         E, H, K, N, Q, R, S, and T. In one embodiment, in both said         first and second heavy chains the amino acid in the position         corresponding to position D265 in a human IgG1 heavy chain is         selected from the group of amino acids consisting of; A, C, F,         G, H, I, L, M, R, T, V, W and Y.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to position D265         in a human heavy chain is selected from the group consisting of;         C, E, H, K, N, Q, R, S, and T.     -   In another embodiment, in both said first and second heavy         chains the amino acid in the position corresponding to position         D265 in a human IgG1 heavy chain is aliphatic uncharged,         aromatic or acidic amino acids.     -   The term “aliphatic uncharged” as used herein in relation to         amino acid residues, refers to any amino acid residue selected         from the group consisting of: A, G, I, L, and V. Thus, in one         embodiment, in both said first and second heavy chains the amino         acid in the position corresponding to position D265 in a human         IgG1 heavy chain is selected from the group consisting of; A, G,         I, L, and V.     -   The term “aromatic” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of: F, T, and W. Thus, in one embodiment, in         both said first and second heavy chains the amino acid in the         position corresponding to position D265 in a human IgG1 heavy         chain is selected from the group consisting of; F, T, and W.     -   The term “acidic” as used herein in relation to amino acid         residues, refers to any amino acid residue chosen from the group         consisting of: D and E. Thus, in one embodiment, in both said         first and second heavy chains the amino acid in the position         corresponding to position D265 in a human IgG1 heavy chain are         selected from the group consisting of; D and E.     -   In a particular embodiment, in both said first and second heavy         chains the amino acid in the position corresponding to position         D265 in a human IgG1 heavy chain is selected from the group         consisting of; A, E, F, G, I, L, T, V, and W.     -   In further embodiment, in at least one of said first and second         heavy chains the amino acid in the position corresponding to         position N297 in a human IgG1 heavy chain, is not N.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acid in the position corresponding to N297 in a         human IgG1 heavy chain, is not N, and the amino acid in the         position corresponding to position P331 in a human IgG1 heavy         chain, is P.     -   In one embodiment, in both said first and second heavy chains         the amino acid in the position corresponding to positions N297         in a human IgG1 heavy chain, is not N.     -   In one embodiment, in both the first and second heavy chains the         amino acid in the position corresponding to N297 in a human IgG1         heavy chain, is not N, and the amino acid in the position         corresponding to position P331 in a human IgG1 heavy chain, is         P.     -   In further embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain, are not L         and L, respectively.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acids in the positions corresponding to L234         and L235 in a human IgG1 heavy chain, are not L and L,         respectively, and the amino acids in the positions corresponding         to positions N297 and P331 in a human IgG1 heavy chain, are N         and P, respectively.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids corresponding to positions L234 and         L235 in a human IgG1 heavy chain are selected from the group         consisting of; A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,         Y, V.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are         hydrophobic or polar amino acids.     -   The term “hydrophobic” as used herein in relation to an amino         acid residue, refers to an amino acid residue selected from the         group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y.         Thus, in one embodiment, in at least one of said first and         second heavy chains the amino acids in the positions         corresponding to positions L234 and L235 in a human IgG1 heavy         chain are each selected from the group consisting of; A, C, F,         G, H, I, M, R, T, V, W, and Y.     -   The term “polar” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in         one embodiment, in at least one of said first and second heavy         chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are each         selected from the group of amino acids consisting of; C, D, E,         H, K, N, Q, R, S, and T.     -   In a particular embodiment, in at least one of said first and         second heavy chains the amino acids in the positions         corresponding to positions L234 and L235 in a human IgG1 heavy         chain are each selected from the group consisting of; A, C, D,         E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to positions L234         and L235 in a human IgG1 heavy chain, are not L and L,         respectively.     -   In one embodiment, in both the first and second heavy chains the         amino acids in the positions corresponding to L234 and L235 in a         human IgG1 heavy chain, are not L and L, respectively, and the         amino acids in the positions corresponding to positions N297 and         P331 in a human IgG1 heavy chain, are N and P, respectively.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to L234 and L235         in a human IgG1 heavy chain are hydrophobic or polar amino         acids.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to positions L234         and L235 in a human IgG1 heavy chain are each selected from the         group consisting of; A, C, F, G, H, I, M, R, T, V, W, and Y.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to positions L234         and L235 in a human IgG1 heavy chain are each selected from the         group of amino acids consisting of; C, D, E, H, K, N, Q, R, S,         and T.     -   In a particular embodiment, in both said first and second heavy         chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are each         selected from the group consisting of; A, C, D, E, F, G, H, I,         K, M, N, Q, R, S, T, V, W, and Y.     -   In another embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are         aliphatic uncharged, aromatic or acidic amino acids.     -   The term “aliphatic uncharged” as used herein in relation to         amino acid residues, refers to any amino acid residue selected         from the group consisting of: A, G, I, L, and V. Thus, in one         embodiment, in at least one of said first and second heavy         chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are each         selected from the group consisting of; A, G, I, and V.     -   The term “aromatic” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of: F, T, and W. Thus, in one embodiment, in at         least one of said first and second heavy chains the amino acids         in the positions corresponding to positions L234 and L235 in a         human IgG1 heavy chain are each selected from the group         consisting of; F, T, and W.     -   The term “acidic” as used herein in relation to amino acid         residues, refers to any amino acid residue chosen from the group         consisting of: D and E. Thus, in one embodiment, in at least one         of said first and second heavy chains the amino acids in the         positions corresponding to positions L234 and L235 in a human         IgG1 heavy chain are each selected from the group consisting of;         D and E.     -   In a particular embodiment, in at least one of said first and         second heavy chains the amino acids in the positions         corresponding to L234 and L235 are each selected from the group         consisting of; A, D, E, F, G, I, T, V, and W.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain, are F and         E; or A and A, respectively.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acids in the positions corresponding to L234         and L235 in a human IgG1 heavy chain, are F and E; or A and A,         respectively, and the amino acids in the positions corresponding         to positions N297 and P331 in a human IgG1 heavy chain, are N         and P, respectively.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to positions L234         and L235 in a human IgG1 heavy chain, are F and E; or A and A,         respectively.     -   In one embodiment, in both the first and second heavy chains the         amino acids in the positions corresponding to L234 and L235 in a         human IgG1 heavy chain, are F and E; or A and A, respectively,         and the amino acids in the positions corresponding to positions         N297 and P331 in a human IgG1 heavy chain, are N and P,         respectively.     -   In a particular embodiment, in at least one of said first and         second heavy chains the amino acids in the positions         corresponding to positions L234 and L235 in a human IgG1 heavy         chain, are F and E, respectively.     -   In one embodiment, in both said first and second heavy chains         the amino acids in the positions corresponding to positions L234         and L235 in a human IgG1 heavy chain, are F and E, respectively.     -   In one embodiment, in at least one of said first and second         heavy chains at least the amino acids in the positions         corresponding to positions L234 and L235 in a human IgG1 heavy         chain, are A and A, respectively.     -   In one embodiment, in both said first and second heavy chains at         least the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain, are A and         A, respectively.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234, L235, and D265 in a human IgG1 heavy chain, are         not L, L, and D, respectively.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acids in the positions corresponding to L234,         L235, and D265 in a human IgG1 heavy chain, are not L, L and D,         respectively, and the amino acids in the positions corresponding         to positions N297 and P331 in a human IgG1 heavy chain, are N         and P, respectively.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids corresponding to positions L234 and         L235 in a human IgG1 heavy chain are selected from the group         consisting of; A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T,         Y, V, and W, and the amino acid corresponding to position D265         is selected from the group consisting of; A, C, E, F, G, H, I,         K, L, M, N, P, Q, R, S, T, Y, V, and W.     -   In one embodiment, in at least one of said first and second         heavy chains the amino acids in the positions corresponding to         positions L234, L235 and D265 in a human IgG1 heavy chain are         hydrophobic or polar amino acids.     -   The term “hydrophobic” as used herein in relation to an amino         acid residue, refers to an amino acid residue selected from the         group consisting of; A, C, F, G, H, I, L, M, R, T, V, W, and Y.         Thus, in one embodiment, in at least one of said first and         second heavy chains the amino acid in the position corresponding         to position D265 in a human IgG1 heavy chain is selected from         the group of amino acids consisting of; A, C, F, G, H, I, L, M,         R, T, V, W and Y, and the amino acids in the positions         corresponding to positions L234 and L235 in a human IgG1 heavy         chain are each selected from the group consisting of; A, C, F,         G, H, I, M, R, T, V, W, and Y.     -   The term “polar” as used herein in relation to amino acid         residues, refers to any amino acid residue selected from the         group consisting of; C, D, E, H, K, N, Q, R, S, and T. Thus, in         one embodiment, in at least one of said first and second heavy         chains the amino acids in the positions corresponding to         positions L234 and L235 in a human IgG1 heavy chain are each         selected from the group of amino acids consisting of; C, D, E,         H, K, N, Q, R, S, and T, the amino acid in the position         corresponding to position D265 in a human heavy chain is         selected from the group consisting of; C, E, H, K, N, Q, R, S,         and T.

In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are hydrophobic or polar amino acids.

In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group of amino acids consisting of; A, C, F, G, H, I, L, M, R, T, V, W and Y, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, C, F, G, H, I, M, R, T, V, W, and Y.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group of amino acids consisting of; C, D, E, H, K, N, Q, R, S, and T, the amino acid in the position corresponding to position D265 in a human heavy chain is selected from the group consisting of; C, E, H, K, N, Q, R, S, and T.

In a particular embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, C, D, E, F, G, H, I, K, M, N, Q, R, S, T, V, W, and Y, and the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, C, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, and Y.

In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

The term “aliphatic uncharged” as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: A, G, I, L, and V. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, G, I, and V.

The term “aromatic” as used herein in relation to amino acid residues, refers to any amino acid residue selected from the group consisting of: F, T, and W. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain are each selected from the group consisting of; F, T, and W.

The term “acidic” as used herein in relation to amino acid residues, refers to any amino acid residue chosen from the group consisting of: D and E. Thus, in one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are each selected from the group consisting of; D and E.

In a particular embodiment, in at least one of said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of; A, D, E, F, G, I, T, V, and W.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235 and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively.

In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are not L, L, and D, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain are aliphatic uncharged, aromatic or acidic amino acids.

In one embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, G, I, L, and V, and the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are each selected from the group consisting of; A, G, I, and V.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are each selected from the group consisting of; D and E.

In a particular embodiment, in both said first and second heavy chains the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of; A, E, F, G, I, L, T, V, and W, and the amino acids in the positions corresponding to L234 and L235 are each selected from the group consisting of; A, D, E, F, G, I, T, V, and W.

In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

In one embodiment, in at least one of the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively.

In one embodiment, in both the first and second heavy chains the amino acids in the positions corresponding to L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A; or A, A, and A, respectively, and the amino acids in the positions corresponding to positions N297 and P331 in a human IgG1 heavy chain, are N and P, respectively.

In a particular embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

In one embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively.

In another embodiment, in at least one of said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

In one embodiment, in both said first and second heavy chains the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain, are F, E, A, Q, and S, respectively.

In one embodiment, the antibody according to the invention, comprises a VH sequence as set out in any one of the sequences in the group of: SEQ ID NOs: 107; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293, a VL sequence as set out in SEQ ID NO:8, and in at least one, or both of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively. Hereby embodiments are provided of anti CD3 antibodies with reduced affinity to human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences as set out in SEQ ID NO:4 and 8, and where the antibodies further comprises a non-activating Fc region.

In a particular embodiment, the antibody according to the invention, comprises a VH sequence as set out in any one of the sequences set out in SEQ ID NOs: 107; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293, a VL sequence as set out in SEQ ID NO:10, and in at least one, or both of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively. Hereby embodiments are provided of anti CD3 antibodies with reduced affinity to human CD3 epsilon compared to a reference antibody comprising the VH and VL sequences as set out in SEQ ID NO:4 and 8, and where the antibodies further comprises a non-activating Fc region and a VL region that allows for enhanced production.

In another embodiment, the antibody according to the invention, comprises a VH sequence as set out in SEQ ID NOs: 221, a VL sequence as set out in SEQ ID NO:8 or 10, and in at least one, or both of the heavy chains the amino acids in positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are F, E, and A, respectively.

In one embodiment of the present invention the human IgG1 heavy chain has the IgG1m(f) sequence as set out in SEQ ID NO:407. In a further embodiment the amino acids in positons corresponding to positions L234, L235, and D265 in a human IgG1m(f) as set out in SEQ ID NO:407, are F, E, and A, respectively.

In one embodiment of the present invention the human IgG1 heavy chain has the IgG1m(f) sequence as set out in SEQ ID NO:409.

In one aspect, the antibody according to the invention comprises the human IgLC2/IgLC3 constant domain lambda light chain of SEQ ID NO:408.

In one aspect, the antibodies according to the invention may be modified in the light chain (LC) and/or heavy chain (HC) to increase the expression level and/or production yield. In one embodiment, the antibodies according to the invention may be modified in the light chain (LC). Such modifications are known in the art and may be performed according to the methods described in e.g. Zheng, L., Goddard, J.-P., Baumann, U., & Reymond, J.-L. (2004). Expression improvement and mechanistic study of the retro-Diels-Alderase catalytic antibody 10F11 by site-directed mutagenesis. Journal of Molecular Biology, 341(3), 807-14. doi:10.1016/j.jmb.2004.06.014.

In one aspect, the antibodies according to the invention may be modified in the VH region and/or the VL region to modify the affinity of the antibodies, such as to reduce or increase the affinity of the antibodies. This may be advantageous in some settings and lead to increased efficacy. In particular low affinity of the CD3 arm may have an impact on the motility of T cells in circulation and at tumor site thus leading to better engagement of T cells with tumor cells, cf. MolhOj et al, Molecular Immunology 44 (2007). In particular this may be useful in bispecific formats, in which a CD3 antibody is used as one of the binding arms. Modifications that lead to reduced antibody affinity are known in the art, see for example Webster et al. Int J Cancer Suppl. 1988; 3:13-6.

Thus in one embodiment the antibody of the present invention comprises a the variable light chain (VL) region comprising the CDR1, CDR2 and CDR3 having the sequences as set forth in SEQ ID NO: 6, GTN, 7 and a variable heavy chain (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 having the CDR sequences selected from one of the groups consisting of;

-   -   a) CDR sequences set forth in SEQ ID NO: 54, 2, 3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequence set forth in SEQ ID NO: 58, 2, 3         [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114N1];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R]; and     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V].

In another aspect, the present invention provides an antibody binding to human CD3, comprising a binding region comprising a variable light chain (VL) region having the sequence set forth in SEQ ID NO 10 and a variable heavy chain (VH) region the CDR1, CDR2 and CDR3 having the sequences selected from one of the groups consisting of:

-   -   a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M];     -   b) CDR1, CDR2 and CDR3 sequence set forth in SEQ ID NO: 58, 2, 3         [T31P];     -   c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106,         3 [N57E];     -   d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114N1];     -   j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R]; and     -   k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V].

Hereby embodiments are provided comprising the T41K mutation in the VL region as set forth in SEQ ID NO:10, thereby allowing increased production of said antibodies.

-   -   In one aspect, the present invention relates to a multispecific         antibody comprising at least a first binding region of an         antibody according to any aspect or embodiment herein described,         and one or more binding regions which binds one or more         different targets than the first binding region. Such a         multispecific antibody may be a bispecific antibody.     -   Thus, in one aspect, the present invention relates to a         bispecific antibody comprising a first binding region of an         antibody according to any aspect or embodiment herein described,         and a second binding region which binds a different target than         the first binding region.     -   The term “multispecific antibody” refers to an antibody having         specificities for at least two different, such as at least         three, typically non-overlapping, epitopes. Such epitopes may be         on the same or different targets. If the epitopes are on         different targets, such targets may be on the same cell or         different cells or cell types.     -   The term “bispecific antibody” refers to an antibody having         specificities for at least two different, typically         non-overlapping, epitopes. Such epitopes may be on the same or         different targets. If the epitopes are on different targets,         such targets may be on the same cell or different cells or cell         types.     -   In one embodiment, the bispecific antibody comprises a first and         a second heavy chain.     -   The embodiments relating to modification of the Fc region and         embodiments relating to specific amino acid substitutions are         contemplated to be part of any bispecific antibody according to         the invention. Thus, in one embodiment, at least one of the         first and second heavy chains comprise one or more amino acids         modified as defined in any embodiment herein described, such as         those described to in relation to providing an inert Fc region.         In one embodiment, both said first and second heavy chains         comprise one or more amino acids modified as defined in any         embodiment herein described, such as those described to in         relation to providing an inert Fc region. Accordingly, the         bispecific antibody comprises an Fc region modified according to         any aspect or embodiment herein described; or at least one of         said first and second heavy chains comprise one or more amino         acids modified as defined in any aspect or embodiment herein         described.     -   Examples of bispecific antibody molecules which may be used in         the present invention comprise (i) a single antibody that has         two arms comprising different antigen-binding regions, (ii) a         single chain antibody that has specificity to two different         epitopes, e.g., via two scFvs linked in tandem by an extra         peptide linker; (iii) a dual-variable-domain antibody (DVD-Ig™),         where each light chain and heavy chain contains two variable         domains in tandem through a short peptide linkage ([40]); (iv) a         chemically-linked bispecific (Fab′)2 fragment; (v) a TandAb®,         which is a fusion of two single chain diabodies resulting in a         tetravalent bispecific antibody that has two binding sites for         each of the target antigens; (vi) a flexibody, which is a         combination of scFvs with a diabody resulting in a multivalent         molecule; (vii) a so called “dock and lock” molecule         (Dock-and-Lock®), based on the “dimerization and docking domain”         in Protein Kinase A, which, when applied to Fabs, can yield a         trivalent bispecific binding protein consisting of two identical         Fab fragments linked to a different Fab fragment; (viii) a         so-called Scorpion molecule, comprising, e.g., two scFvs fused         to both termini of a human Fab-arm; and (ix) a diabody.     -   In one embodiment, the bispecific antibody of the present         invention is a diabody, a cross-body, or a bispecific antibody         obtained via a controlled Fab arm exchange, e.g. DuoBody® (such         as described in [41]) as those described in the present         invention.     -   Examples of different classes of bispecific antibodies include         but are not limited to (i) IgG-like molecules with complementary         CH3 domains to force heterodimerization; (ii) recombinant         IgG-like dual targeting molecules, wherein the two sides of the         molecule each contain the Fab fragment or part of the Fab         fragment of at least two different antibodies; (iii) IgG fusion         molecules, wherein full length IgG antibodies are fused to extra         Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules,         wherein single chain Fv molecules or stabilized diabodies are         fused to heavy-chain constant-domains, Fc-regions or parts         thereof; (v) Fab fusion molecules, wherein different         Fab-fragments are fused together, fused to heavy-chain         constant-domains, Fc-regions or parts thereof; and (vi) ScFv-         and diabody-based and heavy chain antibodies (e.g., domain         antibodies, Nanobodies®) wherein different single chain Fv         molecules or different diabodies or different heavy-chain         antibodies (e.g. domain antibodies, Nanobodies®) are fused to         each other or to another protein or carrier molecule fused to         heavy-chain constant-domains, Fc-regions or parts thereof.     -   Examples of IgG-like molecules with complementary CH3 domains         molecules include but are not limited to the Triomab® (Trion         Pharma/Fresenius Biotech, [42]), the Knobs-into-Holes         (Genentech, [43]), CrossMAbs (Roche, [44]) and the         electrostatically-matched (Amgen, [45]-[46]; Chugai, [47];         Oncomed, [48]), the LUZ-Y (Genentech, Wranik et al. 3. Biol.         Chem. 2012, 287(52): 43331-9, doi: 10.1074/jbc.M112.397869. Epub         2012 Nov. 1), DIG-body and PIG-body (Pharmabcine, WO2010134666,         WO2014081202), the Strand Exchange Engineered Domain body         (SEEDbody)(EMD Serono, [49]), the Biclonics (Merus,         WO2013157953), FcΔAdp (Regeneron, [50]), bispecific IgG1 and         IgG2 (Pfizer/Rinat, [51]), Azymetric scaffold (Zymeworks/Merck,         [52]), mAb-Fv (Xencor, [53]), bivalent bispecific antibodies         (Roche, WO2009080254) and DuoBody® molecules (Genmab A/S, [41]).     -   Examples of recombinant IgG-like dual targeting molecules         include but are not limited to Dual Targeting (DT)-Ig         (GSK/Domantis, WO2009058383), Two-in-one Antibody (Genentech,         Bostrom, et al 2009. Science 323, 1610-1614), Cross-linked Mabs         (Karmanos Cancer Center), mAb2 (F-Star, [54]), Zybodies™         (Zyngenia, LaFleur et al. MAbs. 2013 March-April; 5(2):208-18),         approaches with common light chain (Crucell/Merus, [55]),         KABodies (NovImmune, WO2012023053) and CovX-Body® (CovX/Pfizer,         Doppalapudi, V. R., et al 2007. Bioorg. Med. Chem. Lett.         17,501-506).     -   Examples of IgG fusion molecules include but are not limited to         Dual Variable Domain (DVD)-Ig™ (Abbott, [56]), Dual domain         double head antibodies (Unilever; Sanofi Aventis, [57]),         IgG-like Bispecific (ImClone/Eli Lilly, Lewis et al. Nat         Biotechnol. 2014 February; 32(2):191-8), Ts2Ab (MedImmune/AZ,         Dimasi et al. J Mol Biol. 2009 Oct. 30; 393(3):672-92) and BsAb         (Zymogenetics, WO2010111625), HERCULES (Biogen Idec, [58]), scFv         fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc,         [59]) and TvAb (Roche, [59], [60]).     -   Examples of Fc fusion molecules include but are not limited to         ScFv/Fc Fusions (Academic Institution, Pearce et al Biochem Mol         Biol Int. 1997 September; 42(6):1179-88.), SCORPION (Emergent         BioSolutions/Trubion, Blankenship J W, et al. AACR 100 th Annual         meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625),         Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics,         [62], [63]) and Dual(ScFv)2-Fab (National Research Center for         Antibody Medicine—China).     -   Examples of Fab fusion bispecific antibodies include but are not         limited to F(ab)2 (Medarex/AMGEN), Dual-Action or Bis-Fab         (Genentech), Dock-and-Lock® (DNL) (ImmunoMedics), Bivalent         Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).     -   Examples of ScFv-, diabody-based and domain antibodies include         but are not limited to Bispecific T Cell Engager (BITE®)         (Micromet, Tandem Diabody (Tandab) (Affimed), Dual Affinity         Retargeting Technology (DART™) (MacroGenics), Single-chain         Diabody (Academic, Lawrence FEBS Lett. 1998 Apr. 3;         425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human         Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY         molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010         August; 88(6):667-75), dual targeting Nanobodies® (Ablynx, Hmila         et al., FASEB J. 2010), dual targeting heavy chain only domain         antibodies.     -   It is further contemplated that any monospecific antibody         fulfilling the assay conditions herein described may form the         basis of a bispecific antibody. I.e. a bispecific antibody         wherein one of the binding regions binds CD3 may originate from         any monospecific CD3 antibody tested in the functional assays         and fulfilling the requirements stated herein. Such a bispecific         antibody may be provided by the methods described in [41], which         is hereby incorporated by reference.     -   In one aspect, the bispecific antibody of the invention         comprises a first Fc-region comprising a first CH3 region, and a         second Fc-region comprising a second CH3 region, wherein the         sequences of the first and second CH3 regions are different and         are such that the heterodimeric interaction between said first         and second CH3 regions is stronger than each of the homodimeric         interactions of said first and second CH3 regions. More details         on these interactions and how they can be achieved are provided         in WO2011131746 and WO2013060867 (Genmab), which are hereby         incorporated by reference.     -   Thus, in a particular embodiment, each of said first and second         heavy chain comprises at least a hinge region, a CH2 and CH3         region, wherein in said first heavy chain at least one of the         amino acids in the positions corresponding to a position         selected from the group consisting of T366, L368, K370, D399,         F405, Y407, and K409 in a human IgG1 heavy chain has been         substituted, and in said second heavy chain at least one of the         amino acids in the positions corresponding to a position         selected from the group consisting of T366, L368, K370, D399,         F405, Y407, and K409 in a human IgG1 heavy chain has been         substituted, and wherein said first and said second heavy chains         are not substituted in the same positions. In this context the         term “substituted”, refers to that the amino acid in a specific         amino acid position has been substituted with another naturally         or non-naturally occurring amino acid. Thus, a “substituted”         amino acid in a position corresponding to the position in a         human IgG1 heavy chain means the amino acid at the particular         position is different from the naturally occurring amino acid in         an IgG1 heavy chain.     -   In one embodiment, in said first heavy chain the amino acid in         the position corresponding to K409 in a human IgG1 heavy chain         is not K, L or M, and optionally the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is F, and in         said second heavy chain at least one of the amino acids in the         positions corresponding to a position selected from the group         consisting of; T366, L368, K370, D399, F405, and Y407 in a human         IgG1 heavy chain has been substituted.     -   In one embodiment, in said first heavy chain the amino acid in         the position corresponding to K409 in a human IgG1 heavy chain         is not K, L or M, and in said second heavy chain the amino acid         in the position corresponding to F405 in a human IgG1 heavy         chain is not F and optionally the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is K.     -   In one embodiment, in said first heavy chain, the amino acid in         the position corresponding to F405 in a human IgG1 heavy chain         is not F, R, and G, and in said second heavy chain the amino         acids in the positions corresponding to a position selected form         the group consisting of; T366, L368, K370, D399, Y407, and K409         in a human IgG1 heavy chain has been substituted.     -   In one embodiment, the amino acid in position corresponding to         K409 in a human IgG1 heavy chain is not K, L or M in said first         heavy chain, and the amino acid in position corresponding to         F405 in a human IgG1 heavy chain is not F.     -   In a further embodiment, the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in said         first heavy chain, and the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in said         second heavy chain, or vice versa.     -   Thus, in one embodiment, the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in the         first heavy chain, and the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in the         second heavy chain.     -   In a further embodiment, the humanized or chimeric CD3 antibody         of the invention contains in at least one of the first and         second heavy chain one or more of the inactivating substitutions         as disclosed in any one of the above embodiments, such as L234F,         L235E, and D265A; and that the amino acid in the position         corresponding to F405 is not F. In one embodiment the humanized         or chimeric CD3 antibody of the invention contains in at least         one of the first and second heavy chain one or more of the         inactivating substitutions as disclosed in any one of the above         embodiments, such as L234F, L235E, and D265A; and a further         substitution in the K409 position, such as K409R. In particular,         in one embodiment, the humanized or chimeric CD3 antibody of the         invention contains in both the first and second heavy chain one         or more of the inactivating substitutions as disclosed in any         one of the above embodiments, such as L234F, L235E, and D265A;         and a substitution in the F405 position, such as F405L. In one         embodiment the humanized or chimeric CD3 antibody of the         invention contains in both the first and second heavy chain one         or more of the inactivating substitutions as disclosed in any         one of the above embodiments, such as L234F, L235E, and D265A;         and a further substitution in the K409 position, such as K409R.         Such antibodies are useful for generating a bispecific antibody.     -   Accordingly, in a further embodiment, in at least one of the         first and second heavy chains the amino acids in the positions         corresponding to position L234, L235, and D265 in a human IgG1         heavy chain are F, E, and A, respectively, the amino acid in the         position corresponding to F405 in a human IgG1 heavy chain is L         in the first heavy chain, and the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in the         second heavy chain.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acids in the positions corresponding to L234,         L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E,         A, N, and P respectively, the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in the         first heavy chain, and the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in the         second heavy chain.     -   In an alternative embodiment, in at least one of the first and         second heavy chains the amino acids in the positions         corresponding to position L234, L235, and D265 in a human IgG1         heavy chain are F, E, and A, respectively, the amino acid in the         position corresponding to K409 in a human IgG1 heavy chain is R         in the first heavy chain, and the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in the         second heavy chain.     -   In one embodiment, in at least one of the first and second heavy         chains the amino acids in the positions corresponding to L234,         L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E,         A, N, and P respectively, the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in the         first heavy chain, and the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in the         second heavy chain.     -   In another embodiment, in both the first and second heavy chains         the amino acids in the positions corresponding to position L234,         L235, and D265 in a human IgG1 heavy chain are F, E, and A,         respectively, the amino acid in the position corresponding to         F405 in a human IgG1 heavy chain is L in the first heavy chain,         and the amino acid in the position corresponding to K409 in a         human IgG1 heavy chain is R in the second heavy chain.     -   In one embodiment, in both the first and second heavy chains the         amino acids in the positions corresponding to L234, L235, D265,         N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P         respectively, the amino acid in the position corresponding to         F405 in a human IgG1 heavy chain is L in the first heavy chain,         and the amino acid in the position corresponding to K409 in a         human IgG1 heavy chain is R in the second heavy chain.     -   In an alternative embodiment, in both the first and second heavy         chains the amino acids in the positions corresponding to         position L234, L235, and D265 in a human IgG1 heavy chain are F,         E, and A, respectively, the amino acid in the position         corresponding to K409 in a human IgG1 heavy chain is R in the         first heavy chain, and the amino acid in the position         corresponding to F405 in a human IgG1 heavy chain is L in the         second heavy chain.     -   In one embodiment, in both the first and second heavy chains the         amino acids in the positions corresponding to L234, L235, D265,         N297, and P331 in a human IgG1 heavy chain are F, E, A, N, and P         respectively, the amino acid in the position corresponding to         K409 in a human IgG1 heavy chain is R in the first heavy chain,         and the amino acid in the position corresponding to F405 in a         human IgG1 heavy chain is L in the second heavy chain.     -   As described herein, T cell recruitment to specific target         cells, such as cancer or tumor cells, provides a way of killing         the target cells. T cell mediated killing may be obtained by a         bispecific antibody targeting CD3 with the first binding region         and another target with the second binding region. Thus, in one         embodiment, the first binding region is according to any         embodiments described herein for the humanized or chimeric CD3         antibody, and the second binding region binds a different target         than the first binding region. It is to be understood that when         the antibody is a bispecific antibody, at least one half of the         antibody, i.e. one of the pair of heavy and light chains of the         antibody, is a humanized or chimeric antibody as herein         described. Thus, one half of the bispecific antibody is a         humanized or chimeric antibody binding CD3 according to the         present invention and the other half may be humanized, chimeric,         fully non-human or fully human binding a second target. Thus, in         one embodiment, the antibody comprises a first and a second         heavy chain, a first and second light chain, wherein said first         heavy and said first light chains are humanized or chimeric and         are connected via disulfide bridges forming a first binding         region; and said second heavy and light chains are fully human         and are connected via disulfide bridges forming a second binding         region, wherein said first binding region is according to any         aspect or embodiment herein described, and said second binding         region binds a different target. In one embodiment, the antibody         comprises a first and a second heavy chain, a first and second         light chain, wherein said first heavy and said first light         chains are humanized or chimeric and are connected via disulfide         bridges forming a first binding region; and said second heavy         and light chains are humanized or chimeric and are connected via         disulfide bridges forming a second binding region, wherein said         first binding region is according to any aspect or embodiment         herein described, and said second binding region binds a         different epitope of CD3 than said first binding region.     -   The term “disulfide bridges” as used herein refers to the         covalent bond between two Cysteine residues, i.e. said         interaction may also be designated a Cys-Cys interaction.     -   The term “target” as used herein, refers to a molecule to which         the binding region of the antibody according to the invention         binds. When used in the context of the binding of an antibody         the term includes any antigen towards which the raised antibody         is directed.     -   In one particular embodiment, the first heavy and the first         light chains are humanized or chimeric and are connected via         disulfide bridges forming a first binding region; and the second         heavy and light chains are fully human and are connected via         disulfide bridges forming a second binding region, wherein the         first binding region is according to any aspect or embodiment         herein described, and the second binding region binds a         different target; and wherein in at least one of the first and         second heavy chains the amino acids in the positions         corresponding to positions L234, L235, and D265 in a human IgG1         heavy chain, are F, E, and A, respectively.     -   In one particular embodiment, the first heavy and the first         light chains are humanized or chimeric and are connected via         disulfide bridges forming a first binding region; and the second         heavy and light chains are fully human and are connected via         disulfide bridges forming a second binding region, wherein the         first binding region is according to any aspect or embodiment         herein described, and the second binding region binds a         different epitope of CD3 than the first binding region; and         wherein in at least one of the first and second heavy chains the         amino acids in the positions corresponding to positions L234,         L235, and D265 in a human IgG1 heavy chain, are F, E, and A,         respectively.     -   In one particular embodiment, the first heavy and the first         light chains are humanized or chimeric and are connected via         disulfide bridges forming a first binding region; and the second         heavy and light chains are fully human and are connected via         disulfide bridges forming a second binding region, wherein the         first binding region is according to any aspect or embodiment         herein described, and the second binding region binds a         different target; and wherein in both the first and second heavy         chains the amino acids in the positions corresponding to         positions L234, L235, and D265 in a human IgG1 heavy chain, are         F, E, and A, respectively.     -   In one particular embodiment, the first heavy and the first         light chains are humanized or chimeric and are connected via         disulfide bridges forming a first binding region; and the second         heavy and light chains are fully human and are connected via         disulfide bridges forming a second binding region, wherein the         first binding region is according to any aspect or embodiment         herein described, and the second binding region binds a         different epitope of CD3 than the first binding region; and         wherein in both the first and second heavy chains the amino         acids in the positions corresponding to positions L234, L235,         and D265 in a human IgG1 heavy chain, are F, E, and A,         respectively.

In another aspect, the present invention relates to a method of reducing the binding affinity of an antibody binding to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2, 3, which method comprises introducing a mutation in one of the three CDR sequences of the said reference antibody selected from a mutation in one of the positions selected from the group of T31M, T31P, N57, H101, S110 and Y114, wherein the positions are numbered according to the reference sequence of the SEQ ID NO: 4.

The numbering of the amino acids in the VH regions and the positions to be mutated are according to the amino acids in SEQ ID NO: 4. Numbering are according to a direct numerical numbering scheme from the first amino acid to number 125 in the direction from N-terminus to the C-terminus. The numerical numbering of positions corresponding to SEQ ID NO:4 is illustrated in FIG. 2 . Further, The CDR regions have been annotated according to the IMGT definitions.

In one embodiment of the invention the method comprises introducing a T31M or T31P mutation. Position T31 is in accordance to SEQ ID NO:4.

In one embodiment of the invention the method comprises introducing a mutation in the position N57. Position N57 is in accordance to SEQ ID NO:4. In one embodiment the mutation is N57E

In one embodiment of the invention the method comprises introducing a mutation in the position H101. Position H101 is in accordance to SEQ ID NO: 4. In one embodiment the mutation is H101G or H101N.

In one embodiment of the invention the method comprises introducing a mutation in the position Y114. Position Y114 is in accordance to of SEQ ID NO: 4. In one embodiment the mutation is Y114, Y114R or Y114V.

In one embodiment of the invention the method comprises introducing a mutation in the mutation in the VH CDR3 region corresponding to a position selected from the group of H101, S110 and Y114.

In one embodiment of the invention the method comprises introducing a mutation in the VH CDR3 region is selected from the group consisting of H101G, H101N, S110A, S110G, Y114M, Y114R and Y114V.

In one embodiment of the invention the method comprises introducing a mutation, wherein antibody has a binding affinity to human CD3 epsilon peptide with SEQ ID NO: 402 corresponding to a K_(D) value from 1.6×10⁻⁸ M to 9.9×10⁻⁸M or from 1.0×10⁻⁷ to 9.9×10⁻⁷ M as determined by Bio-Layer Interferometry.

In one embodiment of the invention the method comprises introducing a mutation, wherein the antibody has a binding affinity to human CD3 epsilon peptide with SEQ ID NO: 402 corresponding to a K_(D) value from 1.4×10⁻⁸ to 1.0×10⁻⁸ M or from as 9.9×10⁻⁹ to 1×10⁻⁹ M as determined by Bio-Layer Interferometry.

In one embodiments of the present invention the antibody has a binding affinity to human CD3 epsilon peptide with SEQ ID NO: 402 corresponding to a K_(D) value from 1.6×10⁻⁸ M to 9.9×10⁻⁸M or from 1.0×10⁻⁷ to 9.9×10⁻⁷ M as determined by Bio-Layer Interferometry.

In another aspect, the present invention relates to a method of increasing the binding affinity of an antibody binding to human CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2, 3, which method comprises introducing a mutation in the VH CDR3 corresponding to position G105, wherein the position is numbered according to the reference sequence of the SEQ ID NO: 4.

In one embodiment of the invention the method comprises introducing a mutation in the position G105. Position G105 is in accordance to SEQ ID NO: 4. In one embodiment the mutation is G105P.

In one embodiment of the invention the method comprises introducing at most 5 further mutations, at most 4 further mutations, at most 3 further mutations, at most 2 further mutations or at most 1 further mutation into the CDRs of the VH region of the reference antibody as set forth in SEQ ID NO: 1, 2, 3.

In one embodiment of the invention the method of increased or reduced binding affinity comprises a binding region comprising a heavy chain variable (VH) region, wherein said VH region comprises the CDR1, CDR2, and CDR3 sequences selected from the group consisting of;

-   -   a) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 54, 2,         3 [T31M]     -   b) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 58, 2,         3 [T31P];     -   c) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1,         106, 3 [N57E];     -   d) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         176 [H101G];     -   e) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         184 [H101N];     -   f) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         220 [G105P];     -   g) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         236 [S110A];     -   h) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         244 [S110G];     -   i) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         284 [Y114M];     -   j) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         292 [Y114R]; and     -   k) CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO: 1, 2,         298 [Y114V].

In another embodiment of the present invention the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In a further embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R or Y114V. In another aspect, the present invention relates to method of reducing or increasing the binding affinity of an antibody to CD3, wherein said antibody comprises a binding region comprising heavy chain variable (VH) region, wherein said VH region comprises a mutation in one of the three CDR sequences of a reference antibody as set forth by CDR1 SEQ ID: 1, CDR2 SEQ ID: 2 and CDR3 SEQ ID: 3, wherein said antibody comprises a mutation in one of the following positions selected from the group of T31M, T31P, N57, H101, G105, S110 and Y114, wherein the positions are corresponding to the reference sequence of the SEQ ID NO: 4.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR1 region sequence corresponding to T31M or T31P. In another embodiment of the present invention the method comprises introducing a mutation in the VH region CDR2 region corresponding to N57E. In a further embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 region corresponding to H101G, H101N, G105P, S110A, S110G, Y114M, Y114R or Y114V.

In further aspect, the present invention relates to a method of reducing the binding affinity of an antibody binding to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein said VH region comprises CDR1, CDR2, and CDR3 having the CDR sequences set forth in SEQ ID NO: 1, 2 and 3, which method comprises introducing a mutation in one of the VH region CDR1, CDR2 or CDR3 sequences as set forth in SEQ ID NO: 1, 2 or 3.

In one embodiment the present invention the method comprises introducing a mutation in one of the three CDR regions of the VH region corresponding to one of the following positions: T31, N57, H101, S110 or Y114, wherein the positions are corresponding to the reference sequence of the SEQ ID NO: 4.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR1 sequence corresponding to position T31, wherein the CDR1 sequence is as set forth in SEQ ID NO 1. When the mutation is represented by X the resulting CDR1 sequence may be presented as GFTFNXYA (SEQ ID NO: 412). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNXYA (SEQ ID NO: 412), CDR2 IRSKYNNYAT (SEQ ID NO: 2) and CDR3 VRHGNFGNSYVSWFAY (SEQ ID NO: 3). In one embodiment the mutation in position T31 in in VH region CDR1 is a T31M or T31P mutation.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR2 sequence corresponding to postion N57, wherein the CDR2 sequence is as set forth in SEQ ID NO 2. When the mutation is represented by X the resulting CDR2 sequence may be presented as IRSKYNXYAT (SEQ ID NO: 413). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNTYA (SEQ ID NO: 1), CDR2 IRSKYNXYAT (SEQ ID NO: 413) and CDR3 VRHGNFGNSYVSWFAY (SEQ ID NO: 3). In one embodiment the mutation in position N57 in VH region CDR2 is a N57E mutation.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 sequence corresponding to postion H101, wherein the CDR3 sequence is as set forth in SEQ ID NO 3. When the mutation is represented by X the resulting CDR3 sequence may be presented as VRXGNFGNSYVSWFAY (SEQ ID NO: 414). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNTYA (SEQ ID NO: 1), CDR2 IRSKYNNYAT (SEQ ID NO: 2) and CDR3 VRXGNFGNSYVSWFAY (SEQ ID NO: 414). In one embodiment the mutation in position H101 in VH region CDR3 is an H101G or an H101N mutation.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 sequence corresponding to postion S110, wherein the CDR3 sequence is as set forth in SEQ ID NO 3. When the mutation is represented by X the resulting CDR3 sequence may be presented as VRHGNFGNSYVXWFAY (SEQ ID NO: 415). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNTYA (SEQ ID NO: 1), CDR2 IRSKYNNYAT (SEQ ID NO: 2) and CDR3 VRHGNFGNSYVXWFAY (SEQ ID NO: 415). In one embodiment the mutation in position H101 in VH region CDR3 is a S110A or a S110G mutation.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 sequence corresponding to position Y114, wherein the CDR3 sequence is as set forth in SEQ ID NO 3. When the mutation is represented by X the resulting CDR3 sequence may be presented as VRHGNFGNSYVSWFAX (SEQ ID NO: 416). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNTYA (SEQ ID NO: 1), CDR2 IRSKYNNYAT (SEQ ID NO: 2) and CDR3 VRHGNFGNSYVSWFAX (SEQ ID NO: 416). In one embodiment the mutation in position Y114 in VH region CDR3 is a Y114M, Y114R or a Y114V mutation.

In one embodiment of the invention the method comprises introducing at most 3 mutations, at most 2 mutations or at most 1 mutation into the one or more of the three CDRs of the VH region of a reference antibody as set forth in SEQ ID NO: 1, 2, 3.

In one embodiment of the invention the method comprises introducing at most 10 mutations, at most 9 mutations at, at most 8 mutations, at most 7 mutations, at most 6 mutations, at most 5 mutations, at most 4 mutations, at most 3 mutations, at most 2 mutations or at most 1 mutation into variable heavy chain frame work region of an antibody, wherein said mutations does preferable not alter binding of the antibody to CD3 compared to the same antibody without the mutation(s).

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR1 sequence selected from T31M or T31P. In another embodiment of the present invention the method comprises introducing a mutation in the VH region CDR2 sequence of N57E. In a further embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 sequence selected from the group of: H101G, H101N, S110A, S110G, Y114M, Y114R and Y114V.

In another aspect, the present invention relates to a method of increasing the binding affinity of an antibody binding to CD3 compared to a reference antibody comprising a heavy chain variable (VH) region, wherein said VH region comprises CDR1, CDR2, and CDR3 having the CDR sequences set forth in SEQ ID NO: 1, 2 and 3, which method comprises introducing a mutation in one of the VH region CDR1, CDR2 or CDR3 sequences as set forth in SEQ ID NO: 1, 2 or 3.

In one embodiment of the present invention the method comprises introducing a mutation in the VH region CDR3 sequence corresponding to postion G105, wherein the CDR3 sequence is as set forth in SEQ ID NO 3. When the mutation is represented by X the resulting CDR3 sequence may be presented as VRHGNFXNSYVSWFAY (SEQ ID NO: 417). In one embodiment the three CDR sequences of the VH region may have the following sequences CDR1 GFTFNTYA (SEQ ID NO: 1), CDR2 IRSKYNNYAT (SEQ ID NO: 2) and CDR3 VRXGNFGNSYVSWFAY (SEQ ID NO: 414). In one embodiment the mutation in position G105 in VH region CDR3 is a G105P mutation.

Nucleic Acid Constructs, Expression Vectors, and Host Cells

-   -   In one aspect, the present invention relates to a nucleic acid         construct encoding one or more sequences set out in Table 1.         Thus, the present invention relates to a nucleic acid construct         encoding any one of the sequences set out in SEQ ID NOs: 107;         221; 59; 245; 299; 285; 55; 185; 179; 237; 177 and 293.     -   In a further aspect, the invention relates to nucleic acid         construct encoding a sequence of a humanized or chimeric CD3         antibody according to the present invention, to expression         vectors comprising a nucleic acid construct according to the         present invention, to host cells comprising such expression         vectors, and to methods of producing such an antibody by         culturing such host cells under appropriate conditions whereby         the antibody is produced and, optionally, retrieved. Humanized         CD3 antibodies may also be denoted as “huCD3”.     -   In one embodiment, the invention provides an expression vector         comprising (i) a nucleic acid sequence encoding a heavy chain         sequence of a humanized or chimeric antibody according to the         invention, (ii) a nucleic acid sequence encoding a light chain         sequence of a humanized or chimeric antibody according to the         invention, or (iii) both (i) and (ii). Thus, the expression         vector comprises one or more nucleic acid constructs or nucleic         acid sequences according to any aspect or embodiment herein         described. In one embodiment, the expression vector of the         invention comprises a nucleic acid sequence encoding one or more         of the heavy chain and light chain CDR sequences wherein the VH         CDR sequences are selected from the group consisting of: SEQ ID         NOs.: 12, 2, 3; 14, 2, 3; 16, 2, 3; 18, 2, 3; 20, 2, 3; 22, 2,         3; 24, 2, 3; 26, 2, 3; 28, 2, 3; 30, 2, 3; 32, 2, 3; 34, 2, 3;         36, 2, 3; 38, 2, 3; 40, 2, 3; 42, 2, 3; 44, 2, 3; 46, 2, 3; 48,         2, 3; 50, 2, 3; 52, 2, 3; 54, 2, 3; 56, 2, 3; 58, 2, 3; 60, 2,         3; 62, 2, 3; 64, 2, 3; 66, 2, 3; 68, 2, 3; 70, 2, 3; 72, 2, 3;         74, 2, 3; 76, 2, 3; 78, 2, 3; 80, 2, 3; 82, 2, 3; 84, 2, 3; 86,         2, 3; 88, 2, 3; 90, 2, 3; 92, 2, 3; 94, 2, 3; 96, 2, 3; 98, 2,         3; 1, 100, 3; 1, 102, 3; 1, 104, 3; 1, 106, 3; 1, 108, 3; 1,         110, 3; 1, 112, 3; 1, 114, 3; 1, 116, 3; 1, 118, 3; 1, 120, 3;         1, 122, 3; 1, 124, 3; 1, 126, 3; 1, 128, 3; 1, 130, 3; 1, 132,         3; 1, 134, 3; 1, 136, 3; 1, 138, 3; 1, 140, 3; 1, 142, 3; 1,         144, 3; 1, 146, 3; 1, 148, 3; 1, 150, 3; 1, 152, 3; 1, 154, 3;         1, 156, 3; 1, 158, 3; 1:1, 2, 176; 1, 2, 178; 1, 2, 180; 1, 2,         182; 1, 2, 184; 1, 2, 186; 1, 2, 188; 1, 2, 190; 1, 2, 192; 1,         2, 194; 1, 2, 196; 1, 2, 198; 1, 2, 200; 1, 2, 202; 1, 2, 204;         1, 2, 206; 1, 2, 208; 1, 2, 210; 1, 2, 212; 1, 2, 214; 1, 2,         216; 1, 2, 218; 1, 2, 220; 1, 2, 222; 1, 2, 224; 1, 2, 226; 1,         2, 228; 1, 2, 230; 1, 2, 232; 1, 2, 234; 1, 2, 236; 1, 2, 238;         1, 2, 240; 1, 2, 242; 1, 2, 244; 1, 2, 246; 1, 2, 248; 1, 2,         250; 1, 2, 252; 1, 2, 254; 1, 2, 256; 1, 2, 258; 1, 2, 260; 1,         2, 262; 1, 2, 264; 1, 2, 266; 1, 2, 268; 1, 2, 270; 1, 2, 272;         1, 2, 274; 1, 2, 276; 1, 2, 278; 1, 2, 280; 1, 2, 282; 1, 2,         284; 1, 2, 286; 1, 2, 288; 1, 2, 290; 1, 2, 292; 1, 2, 294; 1,         2, 296; 1, 2, 298 and 1, 2, 300. and wherein the VL CDR         sequences are selected from the group consisting of CDR         sequences as set forth in SEQ ID NO: 6, GTN, 7; 302, GTN, 7;         304, GTN, 7; 306, GTN, 7; 308, GTN, 7; 310, GTN, 7; 312, GTN, 7;         314, GTN, 7; 316, GTN, 7; 318, GTN, 7; 320, GTN, 7; 322, GTN, 7;         324, GTN, 7; 326, GTN, 7; 328, GTN, 7; 330, GTN, 7; 6, GTN, 332;         6, GTN, 334; 6, GTN, 336; 6, GTN, 338; 6, GTN, 340; 6, GTN, 342;         6, GTN, 344; 6, GTN, 346; 6, GTN, 348; 6, GTN, 350; 6, GTN, 352;         6, GTN, 354; 6, GTN, 356; 6, GTN, 358; 6, GTN, 360; 6, GTN, 362;         6, GTN, 364; 6, GTN, 366; 6, GTN, 368; 6, GTN, 370; 6, GTN, 372;         6, GTN, 374; 6, GTN, 376; 6, GTN, 378; 6, GTN, 380; 6, GTN, 382;         6, GTN, 384; GTN, 386; 6, GTN, 388; 6, GTN, 390; GTN, 392; and         6, GTN, 394.     -   In one embodiment, the expression vector of the invention         comprises a nucleic acid sequence encoding one or more of the         heavy chain and light chain CDR sequences wherein VL region         CDR1, CDR2, CDR3 region CDR sequences comprise the CDR sequences         as set forth in SEQ ID NO: 6, GTN, 7 and VH region CDR1, CDR2,         CDR3 region CDR sequences are selected from the group consisting         of: CDR1, CDR2, CDR3 as set forth in SEQ ID NOs.: 54, 2, 3;         CDR1, CDR2, CDR3 as set forth in SEQ ID NO: 58, 2, 3; CDR1,         CDR2, CDR3 as set forth in SEQ ID NO:1, 106, 3; CDR1, CDR2, CDR3         as set forth in SEQ ID NO: 1, 2, 176; CDR1, CDR2, CDR3 as set         forth in SEQ ID NO: 1, 2, 184; CDR1, CDR2, CDR3 as set forth in         SEQ ID NO 1, 2, 220; 1, 2, CDR1, CDR2, CDR3 as set forth in SEQ         ID NO 236; 1, 2, 244; CDR1, CDR2, CDR3 as set forth in SEQ ID NO         1, 2, 284; CDR1, CDR2, CDR3 as set forth in SEQ ID NO 1, 2, 292         and CDR1, CDR2, CDR3 as set forth in SEQ ID N01, 2, 298.     -   In a particular embodiment, the expression vector comprises a         nucleic acid sequence encoding a variant of one or more of the         above amino acid sequences, said variant having at most 25 amino         acid modifications, such as at most 20, such as at most 15, 14,         13, 12, or 11 amino acid modifications, such as 10, 9, 8, 7, 6,         5, 4, 3, 2, or 1 amino acid modifications, such as deletions or         insertions, preferably substitutions, such as conservative or         non-conservative substitutions, or at least 80% identity to any         of said sequences, such as at least 85% identity or 90% identity         or 95% identity, such as 96% identity or 97% identity or 98%         identity or 99% identity to any of the afore-mentioned amino         acid sequences. The present invention also relates to nucleic         acid sequences different from the above mentioned nucleic acid         sequences but which due to the variance of the genetic code         encode the same amino acid sequence as an antibody of the         present invention. E.g. the nucleic acid sequence may vary but         result in an identical amino acid sequences as any amino acid         sequence herein described. It is well-known for the skilled         person how to identify such further nucleic acid sequences based         on the genetic code.     -   In a further embodiment, the expression vector further comprises         a nucleic acid sequence encoding the constant region of a light         chain, a heavy chain or both light and heavy chains of an         antibody, e.g. a human antibody.     -   Such expression vectors as described above may be used for         recombinant production of antibodies of the invention.     -   An expression vector in the context of the present invention may         be any suitable vector, including chromosomal, non-chromosomal,         and synthetic nucleic acid vectors (a nucleic acid sequence         comprising a suitable set of expression control elements).         Examples of such vectors include derivatives of SV40, bacterial         plasmids, phage DNA, baculovirus, yeast plasmids, vectors         derived from combinations of plasmids and phage DNA, and viral         nucleic acid (RNA or DNA) vectors. In one embodiment, a         humanized or chimeric CD3 antibody-encoding nucleic acid is         comprised in a naked DNA or RNA vector, including, for example,         a linear expression element (as described in for instance [64]),         a compacted nucleic acid vector (as described in for instance         [65] and/or [66]), a plasmid vector such as pBR322, pUC 19/18,         or pUC 118/119, a “midge” minimally-sized nucleic acid vector         (as described in for instance [67]), or as a precipitated         nucleic acid vector construct, such as a CaPO₄ ⁻-precipitated         construct (as described in for instance [68], [69], [70], and         [71]). Such nucleic acid vectors and the usage thereof are well         known in the art (see for instance [72] and [73]).     -   In one embodiment, the vector is suitable for expression of the         humanized or chimeric CD3 antibody in a bacterial cell. Examples         of such vectors include expression vectors such as BlueScript         (Stratagene), pIN vectors ([74]), pET vectors (Novagen, Madison         Wis.) and the like.     -   An expression vector may also or alternatively be a vector         suitable for expression in a yeast system. Any vector suitable         for expression in a yeast system may be employed. Suitable         vectors include, for example, vectors comprising constitutive or         inducible promoters such as alpha factor, alcohol oxidase and         PGH (reviewed in: [75] and [76]).     -   A nucleic acid construct and/or vector may also comprise a         nucleic acid sequence encoding a secretion/localization         sequence, which can target a polypeptide, such as a nascent         polypeptide chain, to the periplasmic space or into cell culture         media. Such sequences are known in the art, and include         secretion leader or signal peptides, organelle-targeting         sequences (e. g., nuclear localization sequences, ER retention         signals, mitochondrial transit sequences, chloroplast transit         sequences), membrane localization/anchor sequences (e. g., stop         transfer sequences, GPI anchor sequences), and the like which         are well-known in the art.     -   In an expression vector of the invention, humanized or chimeric         CD3 antibody-encoding nucleic acids may comprise or be         associated with any suitable promoter, enhancer, and other         expression-facilitating elements. Examples of such elements         include strong expression promoters (e. g., human CMV IE         promoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTR         promoters), effective poly (A) termination sequences, an origin         of replication for plasmid product in E. coli, an antibiotic         resistance gene as selectable marker, and/or a convenient         cloning site (e.g., a polylinker). Nucleic acid constructs         and/or vectors may also comprise an inducible promoter as         opposed to a constitutive promoter such as CMV IE (the skilled         person will recognize that such terms are actually descriptors         of a degree of gene expression under certain conditions).     -   In one embodiment, the humanized or chimeric CD3         antibody-encoding expression vector is positioned in and/or         delivered to the host cell or host animal via a viral vector.     -   Such expression vectors may be used for recombinant production         of humanized or chimeric CD3 antibodies.     -   In one aspect, the invention provides a host cell comprising an         expression vector according to the invention.     -   In one aspect, the humanized or chimeric CD3 antibodies of any         aspect or embodiment described herein are provided by use of         recombinant eukaryotic, recombinant prokaryotic, or recombinant         microbial host cell which produces the antibody. Accordingly,         the invention provides a recombinant eukaryotic, recombinant         prokaryotic, or recombinant microbial host cell, which produces         a humanized or chimeric CD3 antibody or immunoglobulin as         defined herein. Examples of host cells include yeast, bacterial         and mammalian cells, such as CHO or HEK-293 cells. For example,         in one embodiment, the host cell comprises a nucleic acid         sequence stably integrated into the cellular genome that         comprises a sequence coding for expression of a humanized or         chimeric CD3 antibody described herein. In another embodiment,         the host cell comprises a non-integrated nucleic acid sequence,         such as a plasmid, cosmid, phagemid, or linear expression         element, which comprises a sequence coding for expression of a         humanized or chimeric CD3 antibody described herein.     -   The term “recombinant host cell” (or simply “host cell”), as         used herein, is intended to refer to a cell into which an         expression vector or nucleic acid construct or sequence has been         introduced. It should be understood that such terms are intended         to refer not only to the particular subject cell, but also to         the progeny of such a cell. Because certain modifications may         occur in succeeding generations due to either mutation or         environmental influences, such progeny may not, in fact, be         identical to the parent cell, but are still included within the         scope of the term “host cell” as used herein. Recombinant host         cells include, for example, eukaryotic host cells, such as CHO         cells, HEK-293 cells, PER.C6, NS0 cells, and lymphocytic cells,         and prokaryotic cells such as E. coli and other eukaryotic hosts         such as plant cells and fungi.     -   In a further aspect, the invention relates to a method for         producing a humanized or chimeric CD3 antibody of the invention,         said method comprising the steps of     -   a) culturing a host cell of the invention as described herein         above, and     -   b) retrieving and/or purifying the antibody of the invention         from the culture media.     -   In a further aspect, the nucleotide sequence encoding a sequence         of a humanized or chimeric CD3 antibody further encodes a second         moiety, such as a therapeutic polypeptide. Exemplary therapeutic         polypeptides are described elsewhere herein. In one embodiment,         the invention relates to a method for producing a humanized or         chimeric CD3 antibody fusion protein, said method comprising the         steps of     -   a) culturing a host cell comprising an expression vector         comprising such a nucleotide sequence, and     -   b) retrieving and/or purifying the humanized or chimeric CD3         antibody fusion protein from the culture media.

Compositions

-   -   In one aspect, the invention provides a composition comprising         the antibody or bispecific antibody according to any aspect and         embodiment herein described.     -   In one aspect, the invention provides a pharmaceutical         composition comprising the antibody or bispecific antibody as         defined in any one of the aspects and embodiments herein         described, and a pharmaceutically acceptable carrier.     -   The pharmaceutical compositions may be formulated with         pharmaceutically acceptable carriers or diluents as well as any         other known adjuvants and excipients in accordance with         conventional techniques such as those disclosed in [77].     -   The pharmaceutically acceptable carriers or diluents as well as         any other known adjuvants and excipients should be suitable for         the humanized or chimeric antibody of the present invention and         the chosen mode of administration. Suitability for carriers and         other components of pharmaceutical compositions is determined         based on the lack of significant negative impact on the desired         biological properties of the chosen compound or pharmaceutical         composition of the present invention (e.g., less than a         substantial impact (10% or less relative inhibition, 5% or less         relative inhibition, etc.)) on antigen binding.     -   A pharmaceutical composition of the present invention may also         include diluents, fillers, salts, buffers, detergents (e. g., a         nonionic detergent, such as Tween-20 or Tween-80), stabilizers         (e.g., sugars or protein-free amino acids), preservatives,         tissue fixatives, solubilizers, and/or other materials suitable         for inclusion in a pharmaceutical composition.     -   The actual dosage levels of the active ingredients in the         pharmaceutical compositions of the present invention may be         varied so as to obtain an amount of the active ingredient which         is effective to achieve the desired therapeutic response for a         particular patient, composition, and mode of administration,         without being toxic to the patient. The selected dosage level         will depend upon a variety of pharmacokinetic factors including         the activity of the particular compositions of the present         invention employed, or the amide thereof, the route of         administration, the time of administration, the rate of         excretion of the particular compound being employed, the         duration of the treatment, other drugs, compounds and/or         materials used in combination with the particular compositions         employed, the age, sex, weight, condition, general health and         prior medical history of the patient being treated, and like         factors well known in the medical arts.     -   The pharmaceutical composition may be administered by any         suitable route and mode. Suitable routes of administering a         humanized or chimeric antibody of the present invention in vivo         and in vitro are well known in the art and may be selected by         those of ordinary skill in the art.     -   In one embodiment, a pharmaceutical composition of the present         invention is administered parenterally.     -   The phrases “parenteral administration” and “administered         parenterally” as used herein means modes of administration other         than enteral and topical administration, usually by injection,         and include epidermal, intravenous, intramuscular,         intra-arterial, intrathecal, intracapsular, intra-orbital,         intracardiac, intradermal, intraperitoneal, intratendinous,         transtracheal, subcutaneous, subcuticular, intra-articular,         subcapsular, subarachnoid, intraspinal, intracranial,         intrathoracic, epidural and intrasternal injection and infusion.     -   In one embodiment that pharmaceutical composition is         administered by intravenous or subcutaneous injection or         infusion.

In a preferred embodiment the pharmaceutical compostions is administered subcutaneous.

-   -   Pharmaceutically acceptable carriers include any and all         suitable solvents, dispersion media, coatings, antibacterial and         antifungal agents, isotonicity agents, antioxidants and         absorption-delaying agents, and the like that are         physiologically compatible with a humanized or chimeric antibody         of the present invention.     -   Examples of suitable aqueous and nonaqueous carriers which may         be employed in the pharmaceutical compositions of the present         invention include water, saline, phosphate buffered saline,         ethanol, dextrose, polyols (such as glycerol, propylene glycol,         polyethylene glycol, and the like), and suitable mixtures         thereof, vegetable oils, such as olive oil, corn oil, peanut         oil, cottonseed oil, and sesame oil, carboxymethyl cellulose         colloidal solutions, tragacanth gum and injectable organic         esters, such as ethyl oleate, and/or various buffers. Other         carriers are well known in the pharmaceutical arts.     -   Pharmaceutically acceptable carriers include sterile aqueous         solutions or dispersions and sterile powders for the         extemporaneous preparation of sterile injectable solutions or         dispersion. The use of such media and agents for         pharmaceutically active substances is known in the art. Except         insofar as any conventional media or agent is incompatible with         the active compound, use thereof in the pharmaceutical         compositions of the present invention is contemplated. When         referring to the “active compound” it is contemplated to also         refer to the humanized or chimeric antibody according to the         present invention.     -   Proper fluidity may be maintained, for example, by the use of         coating materials, such as lecithin, by the maintenance of the         required particle size in the case of dispersions, and by the         use of surfactants.     -   Pharmaceutical compositions of the present invention may also         comprise pharmaceutically acceptable antioxidants for         instance (1) water-soluble antioxidants, such as ascorbic acid,         cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,         sodium sulfite and the like; (2) oil-soluble antioxidants, such         as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated         hydroxytoluene (BHT), lecithin, propyl gallate,         alpha-tocopherol, and the like; and (3) metal-chelating agents,         such as citric acid, ethylenediamine tetraacetic acid (EDTA),         sorbitol, tartaric acid, phosphoric acid, and the like.     -   Pharmaceutical compositions of the present invention may also         comprise isotonicity agents, such as sugars, polyalcohols, such         as mannitol, sorbitol, glycerol or sodium chloride in the         compositions.     -   The pharmaceutical compositions of the present invention may         also contain one or more adjuvants appropriate for the chosen         route of administration such as preservatives, wetting agents,         emulsifying agents, dispersing agents, preservatives or buffers,         which may enhance the shelf life or effectiveness of the         pharmaceutical composition. The humanized or chimeric antibody         of the present invention may be prepared with carriers that will         protect the compound against rapid release, such as a controlled         release formulation, including implants, transdermal patches,         and micro-encapsulated delivery systems. Such carriers may         include gelatin, glyceryl monostearate, glyceryl distearate,         biodegradable, biocompatible polymers such as ethylene vinyl         acetate, polyanhydrides, polyglycolic acid, collagen,         poly-orthoesters, and polylactic acid alone or with a wax, or         other materials well known in the art. Methods for the         preparation of such formulations are generally known to those         skilled in the art (see e.g., [78]).     -   In one embodiment, the humanized or chimeric antibody of the         present invention may be formulated to ensure proper         distribution in vivo. Pharmaceutically acceptable carriers for         parenteral administration include sterile aqueous solutions or         dispersions and sterile powders for the extemporaneous         preparation of sterile injectable solutions or dispersion. The         use of such media and agents for pharmaceutically active         substances is known in the art. Except insofar as any         conventional media or agent is incompatible with the active         compound, use thereof in the pharmaceutical compositions of the         present invention is contemplated. Other active or therapeutic         compounds may also be incorporated into the compositions.     -   Pharmaceutical compositions for injection must typically be         sterile and stable under the conditions of manufacture and         storage. The composition may be formulated as a solution,         micro-emulsion, liposome, or other ordered structure suitable to         high drug concentration. The carrier may be an aqueous or a         non-aqueous solvent or dispersion medium containing for instance         water, ethanol, polyols (such as glycerol, propylene glycol,         polyethylene glycol, and the like), and suitable mixtures         thereof, vegetable oils, such as olive oil, and injectable         organic esters, such as ethyl oleate. The proper fluidity may be         maintained, for example, by the use of a coating such as         lecithin, by the maintenance of the required particle size in         the case of dispersion and by the use of surfactants. In many         cases, it will be preferable to include isotonic agents, for         example, sugars, polyalcohols such as glycerol, mannitol,         sorbitol, or sodium chloride in the composition. Prolonged         absorption of the injectable compositions may be brought about         by including in the composition an agent that delays absorption,         for example, monostearate salts and gelatin. Sterile injectable         solutions may be prepared by incorporating the active compound         in the required amount in an appropriate solvent with one or a         combination of ingredients e.g. as enumerated above, as         required, followed by sterilization microfiltration. Generally,         dispersions are prepared by incorporating the active compound         into a sterile vehicle that contains a basic dispersion medium         and the required other ingredients e.g. from those enumerated         above. In the case of sterile powders for the preparation of         sterile injectable solutions, examples of methods of preparation         are vacuum drying and freeze-drying (lyophilization) that yield         a powder of the active ingredient plus any additional desired         ingredient from a previously sterile-filtered solution thereof.     -   Sterile injectable solutions may be prepared by incorporating         the active compound in the required amount in an appropriate         solvent with one or a combination of ingredients enumerated         above, as required, followed by sterilization microfiltration.         Generally, dispersions are prepared by incorporating the active         compound into a sterile vehicle that contains a basic dispersion         medium and the required other ingredients from those enumerated         above. In the case of sterile powders for the preparation of         sterile injectable solutions, examples of methods of preparation         are vacuum-drying and freeze-drying (lyophilization) that yield         a powder of the active ingredient plus any additional desired         ingredient from a previously sterile-filtered solution thereof.

Therapeutic Applications

-   -   In another aspect, the present invention relates to a humanized         or chimeric antibody, or pharmaceutical composition of the         invention as defined in any aspect or embodiment herein         described, for use as a medicament.     -   In another aspect, the present invention relates to a humanized         or chimeric antibody, or pharmaceutical composition of the         invention as defined in any aspect or embodiment herein         described, for use in the treatment of a disease.     -   In one embodiment of the present invention a bispecific         antibody, a composition, a pharmaceutical composition, for use         in the treatment of a disease.     -   In one embodiment of the present invention, bispecific antibody,         a composition, a pharmaceutical composition is for use for the         treatment of a disease, wherein the disease is cancer, an         infectious disease, or autoimmune diseases.     -   The humanized or chimeric antibody or pharmaceutical composition         of the invention can be used as in the treatment of any cancer         wherein the effector mechanisms of cytotoxic T cells are         desired. For example, the humanized or chimeric antibody may be         administered to cells in culture, e.g., in vitro or ex vivo, or         to human subjects, e.g. in vivo, to treat or prevent disorders         such as cancer, inflammatory or autoimmune disorders. As used         herein, the term “subject” is typically a human which respond to         the humanized or chimeric antibody, or pharmaceutical         composition. Subjects may for instance include human patients         having disorders that may be corrected or ameliorated by         modulating a target function or by leading to killing of the         cell, directly or indirectly.     -   In another aspect, the present invention provides methods for         treating or preventing a disorder, such as cancer, wherein         recruitment of T cells would contribute to the treatment or         prevention, which method comprises administration of a         therapeutically effective amount of a humanized or chimeric         antibody, or pharmaceutical composition of the present invention         to a subject in need thereof. The method typically involves         administering to a subject a humanized or chimeric antibody in         an amount effective to treat or prevent the disorder.     -   In one particular aspect, the present invention relates to a         method of treatment of cancer comprising administering the         humanized or chimeric antibody or pharmaceutical composition of         the invention as defined in any aspect and embodiments herein         described, to a subject in need thereof.     -   In another aspect, the present invention relates to the use or         the method as defined in any aspect or embodiments herein         described wherein the humanized or chimeric antibody is a         bispecific antibody specifically binding to both CD3 and a         cancer-specific target, or a target that is overexpressed in         cancer or associated with cancer, such as HER2, CD19, EpCAM,         EGFR, CD66e (or CEA, CEACAM5), CD33, EphA2 or MCSP (or HMW-MAA),         CD20 and wherein the disease is cancer, such as breast cancer,         prostate cancer, non-small cell lung cancer, bladder cancer,         ovarian cancer, gastric cancer, colorectal cancer, esophageal         cancer and squamous cell carcinoma of the head & neck, cervical         cancer, pancreatic cancer, testis cancer, malignant melanoma, a         soft-tissue cancer (e.g., synovial sarcoma), an indolent or         aggressive form of B-cell lymphoma, chronic lymphatic leukemia         or acute lymphatic leukemia.     -   The efficient dosages and dosage regimens for the humanized or         chimeric antibody depend on the disease or condition to be         treated and may be determined by the persons skilled in the art.     -   A physician having ordinary skill in the art may readily         determine and prescribe the effective amount of the         pharmaceutical composition required. For example, the physician         could start doses of the humanized or chimeric antibody employed         in the pharmaceutical composition at levels lower than that         required in order to achieve the desired therapeutic effect and         gradually increase the dosage until the desired effect is         achieved. In general, a suitable dose of a composition of the         present invention will be that amount of the humanized or         chimeric antibody which is the lowest dose effective to produce         a therapeutic effect according to a particular dosage regimen.         Such an effective dose will generally depend upon the factors         described above.     -   For example, an “effective amount” for therapeutic use may be         measured by its ability to stabilize the progression of disease.         The ability of a compound to inhibit cancer may, for example, be         evaluated in an animal model system predictive of efficacy in         human tumors. Alternatively, this property of a composition may         be evaluated by examining the ability of the humanized or         chimeric antibody to inhibit cell growth or to induce         cytotoxicity by in vitro assays known to the skilled         practitioner. A therapeutically effective amount of a         therapeutic compound, i.e. a therapeutic humanized or chimeric         antibody, or pharmaceutical composition according to the         invention, may decrease tumor size, or otherwise ameliorate         symptoms in a subject. One of ordinary skill in the art would be         able to determine such amounts based on such factors as the         subject's size, the severity of the subject's symptoms, and the         particular composition or route of administration selected.     -   An exemplary, non-limiting range for a therapeutically effective         amount of a humanized or chimeric antibody of the invention is         about 0.001-30 mg/kg, such as about 0.001-20 mg/kg, such as         about 0.001-10 mg/kg, such as about 0.001-5 mg/kg, for example         about 0.001-2 mg/kg, such as about 0.001-1 mg/kg, for instance         about 0.001, about 0.01, about 0.1, about 1, about 5, about 8,         about 10, about 12, about 15, about 18 mg/kg.     -   Administration may e.g. be intravenous, intramuscular,         intraperitoneal, or subcutaneous, and for instance administered         proximal to the site of the target.     -   Dosage regimens in the above methods of treatment and uses are         adjusted to provide the optimum desired response (e.g., a         therapeutic response). For example, a single bolus may be         administered, several divided doses may be administered over         time or the dose may be proportionally reduced or increased as         indicated by the exigencies of the therapeutic situation.     -   In one embodiment, the efficacy of the treatment is monitored         during the therapy, e.g. at predefined points in time.     -   If desired, an effective daily dose of a pharmaceutical         composition may be administered as two, three, four, five, six         or more sub-doses administered separately at appropriate         intervals throughout the day, optionally, in unit dosage forms.         In another embodiment, the humanized or chimeric antibody, or         pharmaceutical composition is administered by slow continuous         infusion over a long period, such as more than 24 hours, in         order to minimize any unwanted side effects.     -   While it is possible for a humanized or chimeric antibody of the         present invention to be administered alone, it is preferable to         administer the humanized or chimeric antibody as a         pharmaceutical composition as described above.     -   An effective dose of a humanized or chimeric antibody of the         invention may also be administered using a weekly, biweekly or         triweekly dosing period. The dosing period may be restricted to,         e.g., 8 weeks, 12 weeks or until clinical progression has been         established. Alternatively, an effective dose of a humanized or         chimeric antibody of the invention may be administered every         second, third or fourth week.     -   In one embodiment, the humanized or chimeric antibody may be         administered by infusion in a weekly dosage of calculated by         mg/m². Such dosages can, for example, be based on the mg/kg         dosages provided above according to the following: dose         (mg/kg)×70:1.8. Such administration may be repeated, e.g., 1 to         8 times, such as 3 to 5 times. The administration may be         performed by continuous infusion over a period of from 2 to 24         hours, such as of from 2 to 12 hours. In one embodiment, the         humanized or chimeric antibody may be administered by slow         continuous infusion over a long period, such as more than 24         hours, in order to reduce toxic side effects.     -   In one embodiment, the humanized or chimeric antibody may be         administered in a weekly dosage of calculated as a fixed dose         for up to 8 times, such as from 4 to 6 times when given once a         week. Such regimen may be repeated one or more times as         necessary, for example, after 6 months or 12 months. Such fixed         dosages can, for example, be based on the mg/kg dosages provided         above, with a body weight estimate of 70 kg. The dosage may be         determined or adjusted by measuring the amount of humanized or         chimeric antibody of the present invention in the blood upon         administration by for instance taking out a biological sample         and using anti-idiotypic antibodies which target the binding         region of the humanized or chimeric antibodies of the present         invention.     -   In one embodiment, the humanized or chimeric antibody may be         administered by maintenance therapy, such as, e.g., once a week         for a period of 6 months or more.     -   A humanized or chimeric antibody may also be administered         prophylactically in order to reduce the risk of developing         cancer, delay the onset of the occurrence of an event in cancer         progression, and/or reduce the risk of recurrence when a cancer         is in remission.     -   Parenteral compositions may be formulated in dosage unit form         for ease of administration and uniformity of dosage. Dosage unit         form as used herein refers to physically discrete units suited         as unitary dosages for the subjects to be treated; each unit         contains a predetermined quantity of active compound calculated         to produce the desired therapeutic effect in association with         the required pharmaceutical carrier. The specification for the         dosage unit forms of the present invention are dictated by and         directly dependent on (a) the unique characteristics of the         active compound and the particular therapeutic effect to be         achieved, and (b) the limitations inherent in the art of         compounding such an active compound for the treatment of         sensitivity in individuals.     -   A humanized or chimeric antibody may also be administered         prophylactically in order to reduce the risk of developing         cancer, delay the onset of the occurrence of an event in cancer         progression, and/or reduce the risk of recurrence when a cancer         is in remission. This may be especially useful in patients         wherein it is difficult to locate a tumor that is known to be         present due to other biological factors.

Diagnostic Applications

-   -   The humanized or chimeric antibody of the invention may also be         used for diagnostic purposes, using a composition comprising a         humanized or chimeric antibody as described herein. Accordingly,         the invention provides diagnostic methods and compositions using         the humanized or chimeric antibodies described herein. Such         methods and compositions can be used for purely diagnostic         purposes, such as detecting or identifying a disease, as well as         for monitoring of the progress of therapeutic treatments,         monitoring disease progression, assessing status after         treatment, monitoring for recurrence of disease, evaluating risk         of developing a disease, and the like.     -   In one aspect, the present invention relates to a method of         diagnosing a disease characterized by involvement or         accumulation of CD3-expression cells, comprising administering         the humanized or chimeric antibody according to the invention,         the composition according to the invention, or the         pharmaceutically composition according to the invention to a         subject, optionally wherein said humanized or chimeric antibody         is labeled with a detectable agent.     -   In one aspect, the humanized or chimeric antibody of the present         invention is used ex vivo, such as in diagnosing a disease in         which cells expressing a specific target of interest and to         which the humanized or chimeric antibody binds, are indicative         of disease or involved in the pathogenesis, by detecting levels         of the target or levels of cells which express the target of         interest on their cell surface in a sample taken from a patient.         This may be achieved, for example, by contacting the sample to         be tested, optionally along with a control sample, with the         humanized or chimeric antibody according to the invention under         conditions that allow for binding of the antibody to the target.         Complex formation can then be detected (e.g., using an ELISA).         When using a control sample along with the test sample, the         level of humanized or chimeric antibody or antibody-target         complex is analyzed in both samples and a statistically         significant higher level of humanized or chimeric antibody or         antibody-target complex in the test sample indicates a higher         level of the target in the test sample compared with the control         sample.     -   Examples of conventional immunoassays in which humanized or         chimeric antibodies of the present invention can be used         include, without limitation, ELISA, RIA, FACS assays, plasmon         resonance assays, chromatographic assays, tissue         immunohistochemistry, Western blot, and/or immunoprecipitation.     -   Accordingly, in one embodiment, the present invention relates to         a method of diagnosing a disease characterized by involvement or         accumulation of CD3-expressing cells, comprising administering         an antibody, bispecific antibody, composition or pharmaceutical         composition according to any aspect or embodiment herein         described, to a subject, optionally wherein the antibody is         labeled with a detectable label.     -   In one embodiment, the invention relates to a method for         detecting the presence of a target, or a cell expressing the         target, in a sample comprising:         -   contacting the sample with a humanized or chimeric antibody             of the invention under conditions that allow for binding of             the humanized or chimeric antibody to the target in the             sample; and         -   analyzing whether a complex has been formed. Typically, the             sample is a biological sample.     -   In one embodiment, the sample is a tissue sample known or         suspected of containing a specific target and/or cells         expressing the target. For example, in situ detection of the         target expression may be accomplished by removing a histological         specimen from a patient, and providing the humanized or chimeric         antibody of the present invention to such a specimen. The         humanized or chimeric antibody may be provided by applying or by         overlaying the humanized or chimeric antibody to the specimen,         which is then detected using suitable means. It is then possible         to determine not only the presence of the target or         target-expressing cells, but also the distribution of the target         or target-expressing cells in the examined tissue (e.g., in the         context of assessing the spread of cancer cells). Using the         present invention, those of ordinary skill will readily perceive         that any of a wide variety of histological methods (such as         staining procedures) may be modified in order to achieve such in         situ detection.     -   In the above assays, the humanized or chimeric antibody can be         labeled with a detectable substance to allow bound antibody to         be detected. Alternatively, bound (primary) specific humanized         or chimeric antibody may be detected by an antibody which is         labeled with a detectable substance and which binds to the         primary specific humanized or chimeric antibody. Furthermore, in         the above assays, a diagnostic composition comprising an         antibody or bispecific antibody according to any aspect or         embodiments herein described may be used. Thus, in one aspect,         the present invention relates to a diagnostic composition         comprising an antibody or bispecific antibody according to any         aspect or embodiment herein described.     -   The level of target in a sample can also be estimated by a         competition immunoassay utilizing target standards labeled with         a detectable substance and an unlabeled target-specific         humanized or chimeric antibody. In this type of assay, the         biological sample, the labeled target standard(s) and the         target-specific humanized or chimeric antibody are combined, and         the amount of labeled target standard bound to the unlabeled         target-specific humanized or chimeric antibody is determined.         The amount of target in the biological sample is inversely         proportional to the amount of labeled target standard bound to         the target-specific humanized or chimeric antibody.     -   Suitable labels for the target-specific humanized or chimeric         antibody, secondary antibody and/or target standard used in in         vitro diagnostic techniques include, without limitation, various         enzymes, prosthetic groups, fluorescent materials, luminescent         materials, and radioactive materials. Examples of suitable         enzymes include horseradish peroxidase, alkaline phosphatase,         β-galactosidase, and acetylcholinesterase; examples of suitable         prosthetic group complexes include streptavidin/biotin and         avidin/biotin; examples of suitable fluorescent materials         include umbelliferone, fluorescein, fluorescein isothiocyanate,         rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride         and phycoerythrin; an example of a luminescent material includes         luminol; and examples of suitable radioactive material include         ¹²⁵I, ¹³¹I, ³⁵S, and ³H.     -   In one aspect, the target-specific humanized or chimeric         antibody of the invention is used in the in vivo imaging of         target-expressing tissues such as tumors. For in vivo methods,         antibody fragments such as, e.g., (Fab′)2, Fab and Fab′         fragments, are particularly advantageous because of their rapid         distribution kinetics.     -   In vivo imaging can be performed by any suitable technique. For         example, a target-specific humanized or chimeric antibody (e.g.,         an antibody or a fragment) labeled with ⁹⁹Tc, ¹³¹I, ¹¹¹In or         other gamma-ray emitting isotope may be used to image         target-specific antibody accumulation or distribution in         target-expressing tissues such as tumors with a gamma         scintillation camera (e.g., an Elscint Apex 409ECT device),         typically using low-energy, high resolution collimator or a         low-energy all-purpose collimator. Alternatively, labeling with         ⁸⁹Zr, ⁷⁶Br, ¹⁸F or other positron-emitting radionuclide may be         used to image target-specific humanized or chimeric antibody, or         antibody fragment distribution in tumors using positron emission         tomography (PET). The images obtained by the use of such         techniques may be used to assess biodistribution of target in a         patient, mammal, or tissue, for example in the context of using         target as a biomarker for the presence of cancer/tumor cells.         Variations on this technique may include the use of magnetic         resonance imaging (MRI) to improve imaging over gamma camera         techniques. Conventional immunoscintigraphy methods and         principles are described in, e.g., [79], [80], and [81].         Moreover, such images may also, or alternatively, serve as the         basis for surgical techniques to remove tumors. Furthermore,         such in vivo imaging techniques may allow for the identification         and localization of a tumor in a situation where a patient is         identified as having a tumor (due to the presence of other         biomarkers, metastases, etc.), but the tumor cannot be         identified by traditional analytical techniques. All of these         methods are features of the present invention.     -   The in vivo imaging and other diagnostic methods provided by the         present invention are particularly useful in the detection of         micrometastases in a human patient (e.g., a patient not         previously diagnosed with cancer or a patient in a period of         recovery/remission from a cancer).     -   In one embodiment, the present invention provides an in vivo         imaging method wherein a target-specific humanized or chimeric         antibody of the present invention is conjugated to a         detection-promoting radio-opaque agent, the conjugated humanized         or chimeric antibody is administered to a host, such as by         injection into the bloodstream, and the presence and location of         the labeled humanized or chimeric antibody in the host is         assayed. Through this technique and any other diagnostic method         provided herein, the present invention provides a method for         screening for the presence of disease-related cells in a human         patient or a biological sample taken from a human patient and/or         for assessing the distribution of target-specific humanized or         chimeric antibody prior to target-specific ADC therapy.     -   For diagnostic imaging, radioisotopes may be bound to a         target-specific humanized or chimeric antibody either directly         or indirectly by using an intermediary functional group. Useful         intermediary functional groups include chelators, such as         ethylenediaminetetraacetic acid and         diethylenetriaminepentaacetic acid (see for instance [82]).     -   In addition to radioisotopes and radio-opaque agents, diagnostic         methods may be performed using target-specific antibodies that         are conjugated to dyes (such as with the biotin-streptavidin         complex), contrast agents, fluorescent compounds or molecules         and enhancing agents (e.g. paramagnetic ions) for magnetic         resonance imaging (MRI) (see, e.g., [83], which describes MRI         techniques and the preparation of antibodies conjugated to a MRI         enhancing agent). Such diagnostic/detection agents may be         selected from agents for use in MRI, and fluorescent compounds.         In order to load a target-specific humanized or chimeric         antibody with radioactive metals or paramagnetic ions, it may be         necessary to react it with a reagent having a long tail to which         a multiplicity of chelating groups are attached for binding the         ions. Such a tail may be a polymer such as a polylysine,         polysaccharide, or another derivatized or derivatizable chain         having pendant groups to which may be bound chelating groups         such as, e.g., porphyrins, polyamines, crown ethers,         bisthiosemicarbazones, polyoximes, and like groups known to be         useful for this purpose. Chelates may be coupled to         target-specific humanized or chimeric antibodies using standard         chemistries.     -   Thus, the present invention provides a diagnostic         target-specific humanized or chimeric antibody, wherein the         target-specific humanized or chimeric antibody is conjugated to         a contrast agent (such as for magnetic resonance imaging,         computed tomography, or ultrasound contrast-enhancing agent) or         a radionuclide that may be, for example, a gamma-, beta-,         alpha-, Auger electron-, or positron-emitting isotope.     -   In one aspect, the present invention relates to a diagnostic         composition comprising an antibody or bispecific antibody         according to the invention.     -   In a further aspect, the invention relates to a kit for         detecting the presence of target antigen or a cell expressing         the target, in a sample, comprising:     -   a target-specific humanized or chimeric antibody of the         invention; and     -   instructions for use of the kit.     -   Thus, in one aspect, the present invention provides a kit for         detecting the presence of a CD3 antigen, or a cell expressing         CD3, in a sample comprising the steps of;

a) contacting the sample with an antibody or bispecific antibody according to the invention, under conditions that allow for formation of a complex between the antibody or bispecific antibody and CD3; and

b) analyzing whether a complex has been formed.

-   -   In one embodiment, the present invention provides a kit for         diagnosis of cancer comprising a container comprising a         target-specific humanized or chimeric antibody, and one or more         reagents for detecting binding of the target-specific humanized         or chimeric antibody to the target. Reagents may include, for         example, fluorescent tags, enzymatic tags, or other detectable         tags. The reagents may also include secondary or tertiary         antibodies or reagents for enzymatic reactions, wherein the         enzymatic reactions produce a product that may be visualized. In         one embodiment, the present invention provides a diagnostic kit         comprising one or more target-specific humanized or chimeric         antibodies of the present invention in labeled or unlabeled form         in suitable container(s), reagents for the incubations for an         indirect assay, and substrates or derivatizing agents for         detection in such an assay, depending on the nature of the         label. Control reagent(s) and instructions for use also may be         included.     -   Diagnostic kits may also be supplied for use with a         target-specific humanized or chimeric antibody, such as a         labeled target-specific antibody, for the detection of the         presence of the target in a tissue sample or host. In such         diagnostic kits, as well as in kits for therapeutic uses         described elsewhere herein, a target-specific humanized or         chimeric antibody typically may be provided in a lyophilized         form in a container, either alone or in conjunction with         additional antibodies specific for a target cell or peptide.         Typically, a pharmaceutically acceptable carrier (e.g., an inert         diluent) and/or components thereof, such as a Tris, phosphate,         or carbonate buffer, stabilizers, preservatives, biocides, inert         proteins, e.g., serum albumin, or the like, also are included         (typically in a separate container for mixing) and additional         reagents (also typically in separate container(s)). In certain         kits, a secondary antibody capable of binding to the         target-specific humanized or chimeric antibody, which typically         is present in a separate container, is also included. The second         antibody is typically conjugated to a label and formulated in a         manner similar to the target-specific humanized or chimeric         antibody of the present invention. Using the methods described         above and elsewhere herein, target-specific humanized or         chimeric antibodies may be used to define subsets of         cancer/tumor cells and characterize such cells and related tumor         tissues.

Anti-Idiotypic Antibodies

-   -   In a further aspect, the invention relates to an anti-idiotypic         antibody which binds to a humanized or chimeric antibody of the         invention as described herein. In one embodiment the invention         relates to an anti-idiotypic antibody which binds to an antibody         of any one of claims or a bispecific antibody according to the         invention.     -   An anti-idiotypic (Id) antibody is an antibody which recognizes         unique determinants generally associated with the         antigen-binding site of an antibody. An anti-Id antibody may be         prepared by immunizing an animal of the same species and genetic         type as the source of a CD3 monoclonal antibody with the         monoclonal antibody to which an anti-Id is being prepared. The         immunized animal typically can recognize and respond to the         idiotypic determinants of the immunizing antibody by producing         an antibody to these idiotypic determinants (the anti-Id         antibody). Such antibodies are described in for instance U.S.         Pat. No. 4,699,880. Such antibodies are further features of the         present invention.     -   An anti-Id antibody may also be used as an “immunogen” to induce         an immune response in yet another animal, producing a so-called         anti-anti-Id antibody. An anti-anti-Id antibody may be         epitopically identical to the original monoclonal antibody,         which induced the anti-Id antibody. Thus, by using antibodies to         the idiotypic determinants of a monoclonal antibody, it is         possible to identify other clones expressing antibodies of         identical specificity. Anti-Id antibodies may be varied (thereby         producing anti-Id antibody variants) and/or derivatized by any         suitable technique, such as those described elsewhere herein         with respect to CD3-specific antibodies of the present         invention. For example, a monoclonal anti-Id antibody may be         coupled to a carrier such as keyhole limpet hemocyanin (KLH) and         used to immunize BALB/c mice. Sera from these mice typically         will contain anti-anti-Id antibodies that have the binding         properties similar, if not identical, to an original/parent CD3         antibody.

Sequences

TABLE 1 SEQ ID NO: Clone name Sequence SEQ ID NO: 1 VH-huCD3-H1 CDR1 GFTFNTYA SEQ ID NO: 2 VH-huCD3-H1 CDR2 IRSKYNNYAT SEQ ID NO: 3 VH-huCD3-H1 CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 4 VH-huCD3-H1 EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 5 VH-huCD3-H1 GAAGTGAAGCTGGTGGAATCTGGCGGCGGACTGGTGCAGCCTG GCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCACCTTCA ACACCTACGCCATGAACTGGGTGCGCCAGGCCCCTGGCAAAGGC CTGGAATGGGTGGCCCGGATCAGAAGCAAGTACAACAATTACGC CACCTACTACGCCGACAGCGTGAAGGACCGGTTCACCATCAGCC GGGACGACAGCAAGAGCAGCCTGTACCTGCAGATGAACAACCTG AAAACCGAGGACACCGCCATGTACTACTGCGTGCGGCACGGCAA CTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGG CACCCTCGTGACAGTGTCTAGC SEQ ID NO: 6 VL-huCD3-L1 CDR1 TGAVTTSNY VL-huCD3-L1 CDR2 GTN SEQ ID NO: 7 VL-huCD3-L1 CDR3 ALWYSNLWV SEQ ID NO: 8 VL-huCD3-L1 QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQ.TPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 9 VL-huCD3-L1 CAGGCCGTCGTGACCCAGGAACCCAGCTTTTCGTGTCTCCTGGC GGCACCGTGACCCTGACCTGCAGATCTTCTACAGGCGCCGTGACC ACCAGCAACTACGCCAACTGGGTGCAGCAGACACCCGGCCAGGC CTTTAGAGGACTGATCGGCGGCACCAACAAGAGGGCACCTGGCG TGCCAGCCAGATTCAGCGGCAGCCTGATCGGAGATAAGGCCGCC CTGACAATCACTGGCGCCCAGGCTGACGACGAGAGCATCTACTTT TGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCAC CAAGCTGACAGTGCTG SEQ ID NO: 6 VL-huCD3-L1-T41K TGAVTTSNY CDR1 VL-huCD3-L1-T41K GTN CDR2 SEQ ID NO: 7 VL-huCD3-L1-T41K ALWYSNLWV CDR3 SEQ ID NO: 10 VL-huCD3-L1-T41K QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 11 VL-huCD3-L1-T41K CAGGCCGTCGTGACCCAGGAACCCAGCTTTTCCGTGTCTCCTGGC GGCACCGTGACCCTGACCTGCAGATCTTCTACAGGCGCCGTGACC ACCAGCAACTACGCCAACTGGGTGCAGCAGAAGCCCGGCCAGGC CTTTAGAGGACTGATCGGCGGCACCAACAAGAGGGCACCTGGCG TGCCAGCCAGATTCAGCGGCAGCCTGATCGGAGATAAGGCCGCC CTGACAATCACTGGCGCCCAGGCTGACGACGAGAGCATCTACTTT TGCGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCAC CAAGCTGACCGTCCTA SEQ ID NO: 12 HC_N30A CDR1 GFTFATYA SEQ ID NO: 2 HC_N30A CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30A CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 13 HC_N30A EVKLVESGGGLVQPGGSLRLSCAASGFTFATYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 14 HC_N30C CDR1 GFTFCTYA SEQ ID NO: 2 HC_N30C CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30C CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 15 HC_N30C EVKLVESGGGLVQPGGSLRLSCAASGFTFCTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 16 HC_N30D CDR1 GFTFDTYA SEQ ID NO: 2 HC_N30D CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30D CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 17 HC_N30D EVKLVESGGGLVQPGGSLRLSCAASGFTFDTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 18 HC_N30F CDR1 GFTFFTYA SEQ ID NO: 2 HC_N30F CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30F CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 19 HC_N30F EVKLVESGGGLVQPGGSLRLSCAASGFTFFTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 20 HC_N30G CDR1 GFTFGTYA SEQ ID NO: 2 HC_N30G CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30G CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 21 HC_N30G EVKLVESGGGLVQPGGSLRLSCAASGFTFGTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 22 HC_N30H CDR1 GFTFHTYA SEQ ID NO: 2 HC_N30H CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30H CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 23 HC_N30H EVKLVESGGGLVQPGGSLRLSCAASGFTFHTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 24 HC_N30K CDR1 GFTFKTYA SEQ ID NO: 2 HC_N30K CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30K CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 25 HC_N30K EVKLVESGGGLVQPGGSLRLSCAASGFTFKTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 26 HC_N30L CDR1 GFTFLTYA SEQ ID NO: 2 HC_N30L CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30L CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 27 HC_N30L EVKLVESGGGLVQPGGSLRLSCAASGFTFLTYAMNWVRQAPGKGLE WVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDT AMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 28 HC_N30P CDR1 GFTFPTYA SEQ ID NO: 2 HC_N30P CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30P CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 29 HC_N30P EVKLVESGGGLVQPGGSLRLSCAASGFTFPTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 30 HC_N30Q CDR1 GFTFQTYA SEQ ID NO: 2 HC_N30Q CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30Q CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 31 HC_N30Q EVKLVESGGGLVQPGGSLRLSCAASGFTFQTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 32 HC_N30R CDR1 GFTFRTYA SEQ ID NO: 2 HC_N30R CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30R CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 33 HC_N30R EVKLVESGGGLVQPGGSLRLSCAASGFTFRTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 34 HC_N30T CDR1 GFIFIIYA SEQ ID NO: 2 HC_N30T CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30T CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 35 HC_N30T EVKLVESGGGLVQPGGSLRLSCAASGFTFTTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 36 HC_N30V CDR1 GFTFVTYA SEQ ID NO: 2 HC_N30V CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30V CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 37 HC_N30V EVKLVESGGGLVQPGGSLRLSCAASGFTFVTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 38 HC_N30W CDR1 GFTFWTYA SEQ ID NO: 2 HC_N30W CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_N30W CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 39 HC_N30W EVKLVESGGGLVQPGGSLRLSCAASGFTFWTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 40 HC_T31A CDR1 GFTFNAYA SEQ ID NO: 2 HC_T31A CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31A CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 41 HC_T31A EVKLVESGGGLVQPGGSLRLSCAASGFTFNAYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 42 HC_T31C CDR1 GFTFNCYA SEQ ID NO: 2 HC_T31C CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31C CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 43 HC_T31C EVKLVESGGGLVQPGGSLRLSCAASGFTFNCYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 44 HC_T31D CDR1 GFTFNDYA SEQ ID NO: 2 HC_T31D CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31D CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 45 HC_T31D EVKLVESGGGLVQPGGSLRLSCAASGFTFNDYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 46 HC_T31E CDR1 GFTFNEYA SEQ ID NO: 2 HC_T31E CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31E CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 47 HC_T31E EVKLVESGGGLVQPGGSLRLSCAASGFTFNEYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 48 HC_T31F CDR1 GFTFNFYA SEQ ID NO: 2 HC_T31F CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31F CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 49 HC_T31F EVKLVESGGGLVQPGGSLRLSCAASGFTFNFYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 50 HC_T31H CDR1 GFTFNHYA SEQ ID NO: 2 HC_T31H CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31H CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 51 HC_T31H EVKLVESGGGLVQPGGSLRLSCAASGFTFNHYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 52 HC_T31L CDR1 GFTFNLYA SEQ ID NO: 2 HC_T31L CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31L CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 53 HC_T31L EVKLVESGGGLVQPGGSLRLSCAASGFTFNLYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 54 HC_T31M CDR1 GFTFNMYA SEQ ID NO: 2 HC_T31M CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31M CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 55 HC_T31M EVKLVESGGGLVQPGGSLRLSCAASGFTFNMYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 56 HC_T31N CDR1 GFTFNNYA SEQ ID NO: 2 HC_T31N CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31N CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 57 HC_T31N EVKLVESGGGLVQPGGSLRLSCAASGFTFNNYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 58 HC_T31P CDR1 GFTFNPYA SEQ ID NO: 2 HC_T31P CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31P CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 59 HC_T31P EVKLVESGGGLVQPGGSLRLSCAASGFTFNPYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 60 HC_T31Q CDR1 GFTFNQYA SEQ ID NO: 2 HC_T31Q CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31Q CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 61 HC_T31Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNQYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 62 HC_T31W CDR1 GFTFNWYA SEQ ID NO: 2 HC_T31W CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31W CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 63 HC_T31W EVKLVESGGGLVQPGGSLRLSCAASGFTFNWYAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 64 HC_T31Y CDR1 GFTFNYYA SEQ ID NO: 2 HC_T31Y CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_T31Y CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 65 HC_T31Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNYYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 66 HC_Y32A CDR1 GFTFNTAA SEQ ID NO: 2 HC_Y32A CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32A CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 67 HC_Y32A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTAAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 68 HC_Y32C CDR1 GFTFNTCA SEQ ID NO: 2 HC_Y32C CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32C CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 69 HC_Y32C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTCAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 70 HC_Y32F CDR1 GFTFNTFA SEQ ID NO: 2 HC_Y32F CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32F CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 71 HC_Y32F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTFAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 72 HC_Y32G CDR1 GFTFNTGA SEQ ID NO: 2 HC_Y32G CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32G CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 73 HC_Y32G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTGAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 74 HC_Y32H CDR1 GFTFNTHA SEQ ID NO: 2 HC_Y32H CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32H CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 75 HC_Y32H EVKLVESGGGLVQPGGSLRLSCAASGFTFNTHAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 76 HC_Y32I CDR1 GFTFNTIA SEQ ID NO: 2 HC_Y32I CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32I CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 77 HC_Y32I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTIAMNWVRQAPGKGLE WVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDT AMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 78 HC_Y32K CDR1 GFTFNTKA SEQ ID NO: 2 HC_Y32K CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32K CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 79 HC_Y32K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTKAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 80 HC_Y32L CDR1 GFTFNTLA SEQ ID NO: 2 HC_Y32L CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32L CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 81 HC_Y32L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTLAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 82 HC_Y32M CDR1 GFTFNTMA SEQ ID NO: 2 HC_Y32M CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32M CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 83 HC_Y32M EVKLVESGGGLVQPGGSLRLSCAASGFTFNTMAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 84 HC_Y32N CDR1 GFTFNTNA SEQ ID NO: 2 HC_Y32N CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32N CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 85 HC_Y32N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTNAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 86 HC_Y32P CDR1 GFTFNTPA SEQ ID NO: 2 HC_Y32P CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32P CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 87 HC_Y32P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTPAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 88 HC_Y32Q CDR1 GFTFNTQA SEQ ID NO: 2 HC_Y32Q CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32Q CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 89 HC_Y32Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTQAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 90 HC_Y32R CDR1 GFTFNTRA SEQ ID NO: 2 HC_Y32R CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32R CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 91 HC_Y32R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTRAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 92 HC_Y32S CDR1 GFTFNTSA SEQ ID NO: 2 HC_Y32S CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32S CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 93 HC_Y32S EVKLVESGGGLVQPGGSLRLSCAASGFTFNTSAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 94 HC_Y32T CDR1 GFTFNTTA SEQ ID NO: 2 HC_Y32T CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32T CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 95 HC_Y32T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTTAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 96 HC_Y32V CDR1 GFTFNTVA SEQ ID NO: 2 HC_Y32V CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32V CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 97 HC_Y32V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTVAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 98 HC_Y32W CDR1 GFTFNTWA SEQ ID NO: 2 HC_Y32W CDR2 IRSKYNNYAT SEQ ID NO: 3 HC_Y32W CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 99 HC_Y32W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTWAMNWVRQAPGKG LEWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57A CDR1 GFTFNTYA SEQ ID NO: 100 HC_N57A CDR2 IRSKYNAYAT SEQ ID NO: 3 HC_N57A CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 101 HC_N57A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNAYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57C CDR1 GFTFNTYA SEQ ID NO: 102 HC_N57C CDR2 IRSKYNCYAT SEQ ID NO: 3 HC_N57C CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 103 HC_N57C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNCYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57D CDR1 GFTFNTYA SEQ ID NO: 104 HC_N57D CDR2 IRSKYNDYAT SEQ ID NO: 3 HC_N57D CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 105 HC_N57D EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNDYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57E CDR1 GFTFNTYA SEQ ID NO: 106 HC_N57E CDR2 IRSKYNEYAT SEQ ID NO: 3 HC_N57E CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 107 HC_N57E EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNEYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57F CDR1 GFTFNTYA SEQ ID NO: 108 HC_N57F CDR2 IRSKYNFYAT SEQ ID NO: 3 HC_N57F CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 109 HC_N57F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNFYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57G CDR1 GFTFNTYA SEQ ID NO: 110 HC_N57G CDR2 IRSKYNGYAT SEQ ID NO: 3 HC_N57G CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 111 HC_N57G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNGYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57I CDR1 GFTFNTYA SEQ ID NO: 112 HC_N57I CDR2 IRSKYNIYAT SEQ ID NO: 3 HC_N57I CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 113 HC_N57I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNIYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDT AMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57K CDR1 GFTFNTYA SEQ ID NO: 114 HC_N57K CDR2 IRSKYNKYAT SEQ ID NO: 3 HC_N57K CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 115 HC_N57K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNKYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57L CDR1 GFTFNTYA SEQ ID NO: 116 HC_N57L CDR2 IRSKYNLYAT SEQ ID NO: 3 HC_N57L CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 117 HC_N57L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNLYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57M CDR1 GFTFNTYA SEQ ID NO: 118 HC_N57M CDR2 IRSKYNMYAT SEQ ID NO: 3 HC_N57M CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 119 HC_N57M EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNMYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57P CDR1 GFTFNTYA SEQ ID NO: 120 HC_N57P CDR2 IRSKYNPYAT SEQ ID NO: 3 HC_N57P CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 121 HC_N57P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNPYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57Q CDR1 GFTFNTYA SEQ ID NO: 122 HC_N57Q CDR2 IRSKYNQYAT SEQ ID NO: 3 HC_N57Q CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 123 HC_N57Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNQYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57R CDR1 GFTFNTYA SEQ ID NO: 124 HC_N57R CDR2 IRSKYNRYAT SEQ ID NO: 3 HC_N57R CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 125 HC_N57R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNRYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57T CDR1 GFTFNTYA SEQ ID NO: 126 HC_N57T CDR2 IRSKYNTYAT SEQ ID NO: 3 HC_N57T CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 127 HC_N57T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNTYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57V CDR1 GFTFNTYA SEQ ID NO: 128 HC_N57V CDR2 IRSKYNVYAT SEQ ID NO: 3 HC_N57V CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 129 HC_N57V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNVYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57W CDR1 GFTFNTYA SEQ ID NO: 130 HC_N57W CDR2 IRSKYNWYAT SEQ ID NO: 3 HC_N57W CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 131 HC_N57W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNWYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_N57Y CDR1 GFTFNTYA SEQ ID NO: 132 HC_N57Y CDR2 IRSKYNYYAT SEQ ID NO: 3 HC_N57Y CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 133 HC_N57Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNYYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59C CDR1 GFTFNTYA SEQ ID NO: 134 HC_A59C CDR2 IRSKYNNYCT SEQ ID NO: 3 HC_A59C CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 135 HC_A59C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYCTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59D CDR1 GFTFNTYA SEQ ID NO: 136 HC_A59D CDR2 IRSKYNNYDT SEQ ID NO: 3 HC_A59D CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 137 HC_A59D EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYDTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59E CDR1 GFTFNTYA SEQ ID NO: 138 HC_A59E CDR2 IRSKYNNYET SEQ ID NO: 3 HC_A59E CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 139 HC_A59E EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYETYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59F CDR1 GFTFNTYA SEQ ID NO: 140 HC_A59F CDR2 IRSKYNNYFT SEQ ID NO: 3 HC_A59F CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 141 HC_A59F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYFTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59G CDR1 GFTFNTYA SEQ ID NO: 142 HC_A59G CDR2 IRSKYNNYGT SEQ ID NO: 3 HC_A59G CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 143 HC_A59G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYGTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59H CDR1 GFTFNTYA SEQ ID NO: 144 HC_A59H CDR2 IRSKYNNYHT SEQ ID NO: 3 HC_A59H CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 145 HC_A59H EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYHTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59I CDR1 GFTFNTYA SEQ ID NO: 146 HC_A59I CDR2 IRSKYNNYIT SEQ ID NO: 3 HC_A59I CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 147 HC_A59I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYITYYADSVKDRFTISRDDSKSSLYLQMNNLKTEDT AMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59K CDR1 GFTFNTYA SEQ ID NO: 148 HC_A59K CDR2 IRSKYNNYKT SEQ ID NO: 3 HC_A59K CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 149 HC_A59K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYKTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59L CDR1 GFTFNTYA SEQ ID NO: 150 HC_A59L CDR2 IRSKYNNYLT SEQ ID NO: 3 HC_A59L CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 151 HC_A59L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYLTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59M CDR1 GFTFNTYA SEQ ID NO: 152 HC_A59M CDR2 IRSKYNNYMT SEQ ID NO: 3 HC_A59M CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 153 HC_A59M EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYMTYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59N CDR1 GFTFNTYA SEQ ID NO: 154 HC_A59N CDR2 IRSKYNNYNT SEQ ID NO: 3 HC_A59N CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 155 HC_A59N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYNTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59P CDR1 GFTFNTYA SEQ ID NO: 156 HC_A59P CDR2 IRSKYNNYPT SEQ ID NO: 3 HC_A59P CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 157 HC_A59P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYPTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59Q CDR1 GFTFNTYA SEQ ID NO: 158 HC_A59Q CDR2 IRSKYNNYQT SEQ ID NO: 3 HC_A59Q CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 159 HC_A59Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYQTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59R CDR1 GFTFNTYA SEQ ID NQ:160 HC_A59R CDR2 IRSKYNNYRT SEQ ID NO: 3 HC_A59R CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 161 HC_A59R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYRTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59S CDR1 GFTFNTYA SEQ ID NO: 162 HC_A59S CDR2 IRSKYNNYST SEQ ID NO: 3 HC_A59S CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 163 HC_A59S EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYSTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59V CDR1 GFTFNTYA SEQ ID NO: 164 HC_A59V CDR2 IRSKYNNYVT SEQ ID NO: 3 HC_A59V CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 165 HC_A59V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYVTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59W CDR1 GFTFNTYA SEQ ID NO: 166 HC_A59W CDR2 IRSKYNNYWT SEQ ID NO: 3 HC_A59W CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 167 HC_A59W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYWTYYADSVKDRFTISRDDSKSSLYLQMNNLKTE DTAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_A59Y CDR1 GFTFNTYA SEQ ID NO: 168 HC_A59Y CDR2 IRSKYNNYYT SEQ ID NO: 3 HC_A59Y CDR3 VRHGNFGNSYVSWFAY SEQ ID NO: 169 HC_A59Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYYTYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101A CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101A CDR2 IRSKYNNYAT SEQ ID NO: 170 HC_H101A CDR3 VRAGNFGNSYVSWFAY SEQ ID NO: 171 HC_H101A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRAGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101C CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101C CDR2 IRSKYNNYAT SEQ ID NO: 172 HC_H101C CDR3 VRCGNFGNSYVSWFAY SEQ ID NO: 173 HC_H101C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRCGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101F CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101F CDR2 IRSKYNNYAT SEQ ID NO: 174 HC_H101F CDR3 VRFGNFGNSYVSWFAY SEQ ID NO: 175 HC_H101F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRFGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101G CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101G CDR2 IRSKYNNYAT SEQ ID NO: 176 HC_H101G CDR3 VRGGNFGNSYVSWFAY SEQ ID NO: 177 HC_H101G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRGGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101I CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101I CDR2 IRSKYNNYAT SEQ ID NO: 178 HC_H101I CDR3 VRIGNFGNSYVSWFAY SEQ ID NO: 179 HC_H101I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRIGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101K CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101K CDR2 IRSKYNNYAT SEQ ID NO: 180 HC_H101K CDR3 VRKGNFGNSYVSWFAY SEQ ID NO: 181 HC_H101K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRKGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101L CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101L CDR2 IRSKYNNYAT SEQ ID NO: 182 HC_H101L CDR3 VRLGNFGNSYVSWFAY SEQ ID NO: 183 HC_H101L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRLGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101N CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101N CDR2 IRSKYNNYAT SEQ ID NO: 184 HC_H101N CDR3 VRNGNFGNSYVSWFAY SEQ ID NO: 185 HC_H101N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRNGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101P CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101P CDR2 IRSKYNNYAT SEQ ID NO: 186 HC_H101P CDR3 VRPGNFGNSYVSWFAY SEQ ID NO: 187 HC_H101P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRPGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101Q CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101Q CDR2 IRSKYNNYAT SEQ ID NO: 188 HC_H101Q CDR3 VRQGNFGNSYVSWFAY SEQ ID NO: 189 HC_H101Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRQGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101R CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101R CDR2 IRSKYNNYAT SEQ ID NO: 190 HC_H101R CDR3 VRRGNFGNSYVSWFAY SEQ ID NO: 191 HC_H101R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRRGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101S CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101S CDR2 IRSKYNNYAT SEQ ID NO: 192 HC_H101S CDR3 VRSGNFGNSYVSWFAY SEQ ID NO: 193 HC_H101S EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRSGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101T CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101T CDR2 IRSKYNNYAT SEQ ID NO: 194 HC_H101T CDR3 VRTGNFGNSYVSWFAY SEQ ID NO: 195 HC_H101T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRTGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101V CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101V CDR2 IRSKYNNYAT SEQ ID NO: 196 HC_H101V CDR3 VRVGNFGNSYVSWFAY SEQ ID NO: 197 HC_H101V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRVGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101W CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101W CDR2 IRSKYNNYAT SEQ ID NO: 198 HC_H101W CDR3 VRWGNFGNSYVSWFAY SEQ ID NO: 199 HC_H101W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRWGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_H101Y CDR1 GFTFNTYA SEQ ID NO: 2 HC_H101Y CDR2 IRSKYNNYAT SEQ ID NO: 200 HC_H101Y CDR3 VRYGNFGNSYVSWFAY SEQ ID NO: 201 HC_H101Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRYGNFGNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105A CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105A CDR2 IRSKYNNYAT SEQ ID NO: 202 HC_G105A CDR3 VRHGNFANSYVSWFAY SEQ ID NO: 203 HC_G105A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFANSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105C CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105C CDR2 IRSKYNNYAT SEQ ID NO: 204 HC_G105C CDR3 VRHGNFCNSYVSWFAY SEQ ID NO: 205 HC_G105C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFCNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105E CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105E CDR2 IRSKYNNYAT SEQ ID NO: 206 HC_G105E CDR3 VRHGNFENSYVSWFAY SEQ ID NO: 207 HC_G105E EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFENSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105F CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105F CDR2 IRSKYNNYAT SEQ ID NO: 208 HC_G105F CDR3 VRHGNFFNSYVSWFAY SEQ ID NO: 209 HC_G105F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFFNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105H CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105H CDR2 IRSKYNNYAT SEQ ID NO: 210 HC_G105H CDR3 VRHGNFHNSYVSWFAY SEQ ID NO: 211 HC_G105H EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFHNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105I CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105I CDR2 IRSKYNNYAT SEQ ID NO: 212 HC_G105I CDR3 VRHGNFINSYVSWFAY SEQ ID NO: 213 HC_G105I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFINSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105L CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105L CDR2 IRSKYNNYAT SEQ ID NO: 214 HC_G105L CDR3 VRHGNFLNSYVSWFAY SEQ ID NO: 215 HC_G105L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFLNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105M CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105M CDR2 IRSKYNNYAT SEQ ID NO: 216 HC_G105M CDR3 VRHGNFMNSYVSWFAY SEQ ID NO: 217 HC_G105M EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFMNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105N CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105N CDR2 IRSKYNNYAT SEQ ID NO: 218 HC_G105N CDR3 VRHGNFNNSYVSWFAY SEQ ID NO: 219 HC_G105N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFNNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105P CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105P CDR2 IRSKYNNYAT SEQ ID NO: 220 HC_G105P CDR3 VRHGNFPNSYVSWFAY SEQ ID NO: 221 HC_G105P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFPNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105Q CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105Q CDR2 IRSKYNNYAT SEQ ID NO: 222 HC_G105Q CDR3 VRHGNFQNSYVSWFAY SEQ ID NO: 223 HC_G105Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFQNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105R CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105R CDR2 IRSKYNNYAT SEQ ID NO: 224 HC_G105R CDR3 VRHGNFRNSYVSWFAY SEQ ID NO: 225 HC_G105R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFRNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105S CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105S CDR2 IRSKYNNYAT SEQ ID NO: 226 HC_G105S CDR3 VRHGNFSNSYVSWFAY SEQ ID NO: 227 HC_G105S EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFSNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105T CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105T CDR2 IRSKYNNYAT SEQ ID NO: 228 HC_G105T CDR3 VRHGNFTNSYVSWFAY SEQ ID NO: 229 HC_G105T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFTNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105V CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105V CDR2 IRSKYNNYAT SEQ ID NO: 230 HC_G105V CDR3 VRHGNFVNSYVSWFAY SEQ ID NO: 231 HC_G105V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFVNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105W CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105W CDR2 IRSKYNNYAT SEQ ID NO: 232 HC_G105W CDR3 VRHGNFWNSYVSWFAY SEQ ID NO: 233 HC_G105W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFWNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_G105Y CDR1 GFTFNTYA SEQ ID NO: 2 HC_G105Y CDR2 IRSKYNNYAT SEQ ID NO: 234 HC_G105Y CDR3 VRHGNFYNSYVSWFAY SEQ ID NO: 235 HC_G105Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFYNSYVSWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110A CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110A CDR2 IRSKYNNYAT SEQ ID NO: 236 HC_S110A CDR3 VRHGNFGNSYVAWFAY SEQ ID NO: 237 HC_S110A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVAWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110C CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110C CDR2 IRSKYNNYAT SEQ ID NO: 238 HC_S110C CDR3 VRHGNFGNSYVCWFAY SEQ ID NO: 239 HC_S110C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVCWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110E CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110E CDR2 IRSKYNNYAT SEQ ID NO: 240 HC_S110E CDR3 VRHGNFGNSYVEWFAY SEQ ID NO: 241 HC_S110E EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVEWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110F CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110F CDR2 IRSKYNNYAT SEQ ID NO: 242 HC_S110F CDR3 VRHGNFGNSYVFWFAY SEQ ID NO: 243 HC_S110F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVFWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110G CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110G CDR2 IRSKYNNYAT SEQ ID NO: 244 HC_S110G CDR3 VRHGNFGNSYVGWFAY SEQ ID NO: 245 HC_S110G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVGWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110H CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110H CDR2 IRSKYNNYAT SEQ ID NO: 246 HC_S110H CDR3 VRHGNFGNSYVHWFAY SEQ ID NO: 247 HC_S110H EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVHWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110K CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110K CDR2 IRSKYNNYAT SEQ ID NO: 248 HC_S110K CDR3 VRHGNFGNSYVKWFAY SEQ ID NO: 249 HC_S110K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVKWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110L CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110L CDR2 IRSKYNNYAT SEQ ID NO: 250 HC_S110L CDR3 VRHGNFGNSYVLWFAY SEQ ID NO: 251 HC_S110L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVLWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110N CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110N CDR2 IRSKYNNYAT SEQ ID NO: 252 HC_S110N CDR3 VRHGNFGNSYVNWFAY SEQ ID NO: 253 HC_S110N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVNWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110P CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110P CDR2 IRSKYNNYAT SEQ ID NO: 254 HC_S110P CDR3 VRHGNFGNSYVPWFAY SEQ ID NO: 255 HC_S110P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVPWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110Q CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110Q CDR2 IRSKYNNYAT SEQ ID NO: 256 HC_S110Q CDR3 VRHGNFGNSYVQWFAY SEQ ID NO: 257 HC_S110Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVQWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110R CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110R CDR2 IRSKYNNYAT SEQ ID NO: 258 HC_S110R CDR3 VRHGNFGNSYVRWFAY SEQ ID NO: 259 HC_S110R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVRWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110T CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110T CDR2 IRSKYNNYAT SEQ ID NO: 260 HC_S110T CDR3 VRHGNFGNSYVTWFAY SEQ ID NO: 261 HC_S110T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVTWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110W CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110W CDR2 IRSKYNNYAT SEQ ID NO: 262 HC_S110W CDR3 VRHGNFGNSYVWWFAY SEQ ID NO: 263 HC_S110W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVWWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_S110Y CDR1 GFTFNTYA SEQ ID NO: 2 HC_S110Y CDR2 IRSKYNNYAT SEQ ID NO: 264 HC_S110Y CDR3 VRHGNFGNSYVYWFAY SEQ ID NO: 265 HC_S110Y EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVYWFAYWGQGTLVTVSS SEQ ID NO: 1 HC_Y114A CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114A CDR2 IRSKYNNYAT SEQ ID NO: 266 HC_Y114A CDR3 VRHGNFGNSYVSWFAA SEQ ID NO: 267 HC_Y114A EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAAWGQGTLVTVSS SEQ ID NO: 1 HC_Y114C CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114C CDR2 IRSKYNNYAT SEQ ID NO: 268 HC_Y114C CDR3 VRHGNFGNSYVSWFAC SEQ ID NO: 269 HC_Y114C EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFACWGQGTLVTVSS SEQ ID NO: 1 HC_Y114E CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114E CDR2 IRSKYNNYAT SEQ ID NO: 270 HC_Y114E CDR3 VRHGNFGNSYVSWFAE SEQ ID NO: 271 HC_Y114E EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAEWGQGTLVTVSS SEQ ID NO: 1 HC_Y114F CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114F CDR2 IRSKYNNYAT SEQ ID NO: 272 HC_Y114F CDR3 VRHGNFGNSYVSWFAF SEQ ID NO: 273 HC_Y114F EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAFWGQGTLVTVSS SEQ ID NO: 1 HC_Y114G CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114G CDR2 IRSKYNNYAT SEQ ID NO: 274 HC_Y114G CDR3 VRHGNFGNSYVSWFAG SEQ ID NO: 275 HC_Y114G EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAGWGQGTLVTVSS SEQ ID NO: 1 HC_Y114H CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114H CDR2 IRSKYNNYAT SEQ ID NO: 276 HC_Y114H CDR3 VRHGNFGNSYVSWFAH SEQ ID NO: 277 HC_Y114H EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAHWGQGTLVTVSS SEQ ID NO: 1 HC_Y114I CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114I CDR2 IRSKYNNYAT SEQ ID NO: 278 HC_Y114I CDR3 VRHGNFGNSYVSWFAI SEQ ID NO: 279 HC_Y114I EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAIWGQGTLVTVSS SEQ ID NO: 1 HC_Y114K CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114K CDR2 IRSKYNNYAT SEQ ID NO: 280 HC_Y114K CDR3 VRHGNFGNSYVSWFAK SEQ ID NO: 281 HC_Y114K EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAKWGQGTLVTVSS SEQ ID NO: 1 HC_Y114L CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114L CDR2 IRSKYNNYAT SEQ ID NO: 282 HC_Y114L CDR3 VRHGNFGNSYVSWFAL SEQ ID NO: 283 HC_Y114L EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFALWGQGTLVTVSS SEQ ID NO: 1 HC_Y114M CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114M CDR2 IRSKYNNYAT SEQ ID NO: 284 HC_Y114M CDR3 VRHGNFGNSYVSWFAM SEQ ID NO: 285 HC_Y114M EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAMWGQGTLVTVSS SEQ ID NO: 1 HC_Y114N CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114N CDR2 IRSKYNNYAT SEQ ID NO: 286 HC_Y114N CDR3 VRHGNFGNSYVSWFAN SEQ ID NO: 287 HC_Y114N EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFANWGQGTLVTVSS SEQ ID NO: 1 HC_Y114P CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114P CDR2 IRSKYNNYAT SEQ ID NO: 288 HC_Y114P CDR3 VRHGNFGNSYVSWFAP SEQ ID NO: 289 HC_Y114P EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAPWGQGTLVTVSS SEQ ID NO: 1 HC_Y114Q CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114Q CDR2 IRSKYNNYAT SEQ ID NO: 290 HC_Y114Q CDR3 VRHGNFGNSYVSWFAQ SEQ ID NO: 291 HC_Y114Q EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAQWGQGTLVTVSS SEQ ID NO: 1 HC_Y114R CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114R CDR2 IRSKYNNYAT SEQ ID NO: 292 HC_Y114R CDR3 VRHGNFGNSYVSWFAR SEQ ID NO: 293 HC_Y114R EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFARWGQGTLVTVSS SEQ ID NO: 1 HC_Y114S CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114S CDR2 IRSKYNNYAT SEQ ID NO: 294 HC_Y114S CDR3 VRHGNFGNSYVSWFAS SEQ ID NO: 295 HC_Y114S EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFASWGQGTLVTVSS SEQ ID NO: 1 HC_Y114T CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114T CDR2 IRSKYNNYAT SEQ ID NO: 296 HC_Y114T CDR3 VRHGNFGNSYVSWFAT SEQ ID NO: 297 HC_Y114T EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFATWGQGTLVTVSS SEQ ID NO: 1 HC_Y114V CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114V CDR2 IRSKYNNYAT SEQ ID NO: 298 HC_Y114V CDR3 VRHGNFGNSYVSWFAV SEQ ID NO: 299 HC_Y114V EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAVWGQGTLVTVSS SEQ ID NO: 1 HC_Y114W CDR1 GFTFNTYA SEQ ID NO: 2 HC_Y114W CDR2 IRSKYNNYAT SEQ ID NO: 300 HC_Y114W CDR3 VRHGNFGNSYVSWFAW SEQ ID NO: 301 HC_Y114W EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGL EWVARIRSKYNNYATYYADSVKDRFTISRDDSKSSLYLQMNNLKTED TAMYYCVRHGNFGNSYVSWFAWWGQGTLVTVSS SEQ ID NO: 302 LC_T31A CDR1 TGAVTASNY LC_T31A CDR2 GTN SEQ ID NO: 7 LC_T31A CDR3 ALWYSNLWV SEQ ID NO: 303 LC_T31A QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTASNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 304 LC_T31D CDR1 TGAVTDSNY LC_T31D CDR2 GTN SEQ ID NO: 7 LC_T31D CDR3 ALWYSNLWV SEQ ID NO: 305 LC_T31D QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTDSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 306 LC_T31E CDR1 TGAVTESNY LC_T31E CDR2 GTN SEQ ID NO: 7 LC_T31E CDR3 ALWYSNLWV SEQ ID NO: 307 LC_T31E QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTESNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 308 LC_T31F CDR1 TGAVTFSNY LC_T31F CDR2 GTN SEQ ID NO: 7 LC_T31F CDR3 ALWYSNLWV SEQ ID NO: 309 LC_T31F QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTFSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 310 LC_T31G CDR1 TGAVTGSNY LC_T31G CDR2 GTN SEQ ID NO: 7 LC_T31G CDR3 ALWYSNLWV SEQ ID NO: 311 LC_T31G QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTGSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 312 LC_T31H CDR1 TGAVTHSNY LC_T31H CDR2 GTN SEQ ID NO: 7 LC_T31H CDR3 ALWYSNLWV SEQ ID NO: 313 LC_T31H QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTHSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL LC_T31I CDR2 GTN SEQ ID NO: 314 LC_T31K CDR1 TGAVTKSNY LC_T31K CDR2 GTN SEQ ID NO: 7 LC_T31K CDR3 ALWYSNLWV SEQ ID NO: 315 LC_T31K QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTKSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 316 LC_T31L CDR1 TGAVTLSNY LC_T31L CDR2 GTN SEQ ID NO: 7 LC_T31L CDR3 ALWYSNLWV SEQ ID NO: 317 LC_T31L QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTLSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 318 LC_T31M CDR1 TGAVTMSNY LC_T31M CDR2 GTN SEQ ID NO: 7 LC_T31M CDR3 ALWYSNLWV SEQ ID NO: 319 LC_T31M QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTMSNYANWVQQKPGQ AFRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAL WYSNLWVFGGGTKLTVL SEQ ID NO: 320 LC_T31N CDR1 TGAVTNSNY LC_T31N CDR2 GTN SEQ ID NO: 7 LC_T31N CDR3 ALWYSNLWV SEQ ID NO: 321 LC_T31N QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTNSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 322 LC_T31P CDR1 TGAVTPSNY LC_T31P CDR2 GTN SEQ ID NO: 7 LC_T31P CDR3 ALWYSNLWV SEQ ID NO: 323 LC_T31P QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTPSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 324 LC_T31Q CDR1 TGAVTQSNY LC_T31Q CDR2 GTN SEQ ID NO: 7 LC_T31Q CDR3 ALWYSNLWV SEQ ID NO: 325 LC_T31Q QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTQSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 326 LC_T31R CDR1 TGAVTRSNY LC_T31R CDR2 GTN SEQ ID NO: 7 LC_T31R CDR3 ALWYSNLWV SEQ ID NO: 327 LC_T31R QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTRSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL LC_T31S CDR2 GTN SEQ ID NO: 328 LC_T31V CDR1 TGAVTVSNY LC_T31V CDR2 GTN SEQ ID NO: 7 LC_T31V CDR3 ALWYSNLWV SEQ ID NO: 329 LC_T31V QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTVSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 330 LC_T31Y CDR1 TGAVTYSNY LC_T31Y CDR2 GTN SEQ ID NO: 7 LC_T31Y CDR3 ALWYSNLWV SEQ ID NO: 331 LC_T31Y QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTYSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92A CDR1 TGAVTTSNY LC_L92A CDR2 GTN SEQ ID NO: 332 LC_L92A CDR3 AAWYSNLWV SEQ ID NO: 333 LC_L92A QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAA WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92C CDR1 TGAVTTSNY LC_L92C CDR2 GTN SEQ ID NO: 334 LC_L92C CDR3 ACWYSNLWV SEQ ID NO: 335 LC_L92C QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAC WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92D CDR1 TGAVTTSNY LC_L92D CDR2 GTN SEQ ID NO: 336 LC_L92D CDR3 ADWYSNLWV SEQ ID NO: 337 LC_L92D QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAD WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92E CDR1 TGAVTTSNY LC_L92E CDR2 GTN SEQ ID NO: 338 LC_L92E CDR3 AEWYSNLWV SEQ ID NO: 339 LC_L92E QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAEW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92F CDR1 TGAVTTSNY LC_L92F CDR2 GTN SEQ ID NO: 340 LC_L92F CDR3 AFWYSNLWV SEQ ID NO: 341 LC_L92F QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAFW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92G CDR1 TGAVTTSNY LC_L92G CDR2 GTN SEQ ID NO: 342 LC_L92G CDR3 AGWYSNLWV SEQ ID NO: 343 LC_L92G QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAG WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L921 CDR1 TGAVTTSNY LC_L921 CDR2 GTN SEQ ID NO: 344 LC_L921 CDR3 AIWYSNLWV SEQ ID NO: 345 LC_L92I QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAIW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92K CDR1 TGAVTTSNY LC_L92K CDR2 GTN SEQ ID NO: 346 LC_L92K CDR3 AKWYSNLWV SEQ ID NO: 347 LC_L92K QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAK WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92M CDR1 TGAVTTSNY LC_L92M CDR2 GTN SEQ ID NO: 348 LC_L92M CDR3 AMWYSNLWV SEQ ID NO: 349 LC_L92M QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAM WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92N CDR1 TGAVTTSNY LC_L92N CDR2 GTN SEQ ID NO: 350 LC_L92N CDR3 ANWYSNLWV SEQ ID NO: 351 LC_L92N QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAN WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92P CDR1 TGAVTTSNY LC_L92P CDR2 GTN SEQ ID NO: 352 LC_L92P CDR3 APWYSNLWV SEQ ID NO: 353 LC_L92P QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAP WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92R CDR1 TGAVTTSNY LC_L92R CDR2 GTN SEQ ID NO: 354 LC_L92R CDR3 ARWYSNLWV SEQ ID NO: 355 LC_L92R QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAR WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92S CDR1 TGAVTTSNY LC_L92S CDR2 GTN SEQ ID NO: 356 LC_L92S CDR3 ASWYSNLWV SEQ ID NO: 357 LC_L92S QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCASW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92T CDR1 TGAVTTSNY LC_L92T CDR2 GTN SEQ ID NO: 358 LC_L92T CDR3 ATWYSNLWV SEQ ID NO: 359 LC_L92T QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCATW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92V CDR1 TGAVTTSNY LC_L92V CDR2 GTN SEQ ID NO: 360 LC_L92V CDR3 AVWYSNLWV SEQ ID NO: 361 LC_L92V QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAV WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92W CDR1 TGAVTTSNY LC_L92W CDR2 GTN SEQ ID NO: 362 LC_L92W CDR3 AWWYSNLWV SEQ ID NO: 363 LC_L92W QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAW WYSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L92Y CDR1 TGAVTTSNY LC_L92Y CDR2 GTN SEQ ID NO: 364 LC_L92Y CDR3 AYWYSNLWV SEQ ID NO: 365 LC_L92Y QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCAYW YSNLWVFGGGTKLTVL SEQ ID NO: 6 LC_L97D CDR1 TGAVTTSNY LC_L97D CDR2 GTN SEQ ID NO: 366 LC_L97D CDR3 ALWYSNDWV SEQ ID NO: 367 LC_L97D QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNDWVFGGGTKLTVL SEQ ID NO: 6 LC_L97E CDR1 TGAVTTSNY LC_L97E CDR2 GTN SEQ ID NO: 368 LC_L97E CDR3 ALWYSNEWV SEQ ID NO: 369 LC_L97E QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNEWVFGGGTKLTVL SEQ ID NO: 6 LC_L97F CDR1 TGAVTTSNY LC_L97F CDR2 GTN SEQ ID NO: 370 LC_L97F CDR3 ALWYSNFWV SEQ ID NO: 371 LC_L97F QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNFWVFGGGTKLTVL SEQ ID NO: 6 LC_L97G CDR1 TGAVTTSNY LC_L97G CDR2 GTN SEQ ID NO: 372 LC_L97G CDR3 ALWYSNGWV SEQ ID NO: 373 LC_L97G QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNGWVFGGGTKLTVL SEQ ID NO: 6 LC_L97H CDR1 TGAVTTSNY LC_L97H CDR2 GTN SEQ ID NO: 374 LC_L97H CDR3 ALWYSNHWV SEQ ID NO: 375 LC_L97H QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNHWVFGGGTKLTVL SEQ ID NO: 6 LC_L97K CDR1 TGAVTTSNY LC_L97K CDR2 GTN SEQ ID NO: 376 LC_L97K CDR3 ALWYSNKWV SEQ ID NO: 377 LC_L97K QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNKWVFGGGTKLTVL SEQ ID NO: 6 LC_L97M CDR1 TGAVTTSNY LC_L97M CDR2 GTN SEQ ID NO: 378 LC_L97M CDR3 ALWYSNMWV SEQ ID NO: 379 LC_L97M QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNMWVFGGGTKLTVL SEQ ID NO: 6 LC_L97N CDR1 TGAVTTSNY LC_L97N CDR2 GTN SEQ ID NO: 380 LC_L97N CDR3 ALWYSNNWV SEQ ID NO: 381 LC_L97N QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNNWVFGGGTKLTVL SEQ ID NO: 6 LC_L97P CDR1 TGAVTTSNY LC_L97P CDR2 GTN SEQ ID NO: 382 LC_L97P CDR3 ALWYSNPWV SEQ ID NO: 383 LC_L97P QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNPWVFGGGTKLTVL SEQ ID NO: 6 LC_L97Q CDR1 TGAVTTSNY LC_L97Q CDR2 GTN SEQ ID NO: 384 LC_L97Q CDR3 ALWYSNQWV SEQ ID NO: 385 LC_L97Q QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNQWVFGGGTKLTVL LC_L97R CDR2 GTN SEQ ID NO: 6 LC_L97S CDR1 TGAVTTSNY LC_L97S CDR2 GTN SEQ ID NO: 386 LC_L97S CDR3 ALWYSNSWV SEQ ID NO: 387 LC_L97S QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNSWVFGGGTKLTVL SEQ ID NO: 6 LC_L97T CDR1 TGAVTTSNY LC_L97T CDR2 GTN SEQ ID NO: 388 LC_L97T CDR3 ALWYSNTWV SEQ ID NO: 389 LC_L97T QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNTWVFGGGTKLTVL SEQ ID NO: 6 LC_L97V CDR1 TGAVTTSNY LC_L97V CDR2 GTN SEQ ID NO: 390 LC_L97V CDR3 ALWYSNVWV SEQ ID NO: 391 LC_L97V QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNVWVFGGGTKLTVL SEQ ID NO: 6 LC_L97W CDR1 TGAVTTSNY LC_L97W CDR2 GTN SEQ ID NO: 392 LC_L97W CDR3 ALWYSNWWV SEQ ID NO: 393 LC_L97W QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNWWVFGGGTKLTVL SEQ ID NO: 6 LC_L97Y CDR1 TGAVTTSNY LC_L97Y CDR2 GTN SEQ ID NO: 394 LC_L97Y CDR3 ALWYSNYWV SEQ ID NO: 395 LC_L97Y QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQA FRGLIGGTNKRAPGVPARFSGSLIGDKAALTITGAQADDESIYFCALW YSNYWVFGGGTKLTVL SEQ ID NO: 396 Mature huTRA KTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIH GLTSNVNNRMASLAIAEDRKSSTLILHRATLRDAAVYYCILPLAGGTS YGKLTFGQGTILTVHPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ TNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANA FNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK VAGFNLLMTLRLWSS SEQ ID NO: 397 Mature huTRB GVSQSPRYKVAKRGQDVALRCDPISGHVSLFWYQQALGQGPEFLTY FQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQRTQQEDSAVYLCASSL GQAYEQYFGPGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVC UXTGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLS SRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLM AMVKRKDF SEQ ID NO: 398 Mature huCD3ζ FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIY RCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLA LGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLG GNWARNK SEQ ID NO: 399 Mature huCD3ϵ QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGG DEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRA RVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVT RGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI SEQ ID NO: 400 Mature huCD3γ QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTED KKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNA ATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLY QPLKDREDDQYSHLQGNQLRRN SEQ ID NO: 401 Mature huCD37 QSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ ALPPR SEQ ID NO: 402 Mature CD3ϵ27-GSKa QDGNEEMGGITQTPYKVSISGTTVILTGGGGSGGGGSGGGGSEIVL TQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQ GTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGE SEQ ID NO: 403 Mature cyno CD3ϵ QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNK (epsilon) EDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENC MEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGA GGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI SEQ ID NO: 404 Mature rhesus CD3ϵ QDGNEEMGSITQTPYHVSISGTTVILTCSQHLGSEVQWQHNGKNKE (epsilon) DSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENC MEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGA GGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI SEQ ID Parent murine VH EVKLLESGGGLVQPKGSLKLSC AASGFTFNTYAMNWVRQAPGKG NO: 405 LEWVARIRSKYNNYATYYADSV KDRFTISRDDSQSILYLQMNNL KTEDTAMYYCVRHGNFGNSYVS WFAYWGQGTLVTVSA SEQ ID Parent murine VL QAVVTQESALTTSPGETVTLTC RSSTGAVTTSNYANWVQEKPDH NO: 406 LFTGLIGGTNKRAPGVPARFSG SLIGDKAALTITGAQTEDEAIY FCALWYSNLWVFGGGTKLTVL SEQ ID IgG1m(f) heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL NO: 407 chain TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK constant region VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR WSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID Human IgLC2/IgLC3 GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADS NO: 408 constant domain SPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHE GSTVEKTVAPTECS SEQ ID NO: 409 IgGlm(f) heavy ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT chain constant SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD region with KRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT FEA mutations CVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK SEQ ID NO: 410 Primer CMV P f GCTTCGCGATGTACGGGCCAGATATAC (MAR5) SEQ ID NO: 411 TK pA r(MARl) GGATACCCCCTAGAGCCCCAGCTGCGCAGATCTGCTATG GC

The CDR regions have been annotated according to the IMGT definitions.

EXAMPLES Example 1—Generation of Humanized CD3 Antibodies and Non-Activating Antibody Variants

Humanization of CD3 Antibodies

Humanization of a murine CD3 antibody (U.S. Pat. No. 8,236,308, described herein as IgG1-CD3) was performed by Antitope (Cambridge, UK) using their improved version of the germline humanization (CDR-grafting) technology (EP 0 629 240). Using this technology, 1 different VH chain (SEQ ID NO:4) and 2 different VL chains (SEQ ID NO:8, 10) were designed. By combining these 1 VH with the 2 VL chains, 2 different antibodies were generated. The humanized variants are described herein as huCD3. Thus, humanized variants comprising a VH and a VL according to the invention, are described as, e.g., IgG1-huCD3-H1L1 meaning that said specific variant is of the IgG1 isotype, is a humanized CD3 and comprises the VH amino acid sequence termed “H1” and is defined according to SEQ ID NO:4, and the VL amino acid sequence termed “L1” and is defined according to SEQ ID NO: 8. Thus, H1 refers to the variable heavy chain region VH1, L1 refers to the variable light chain region VL1, and so forth.

-   -   In particular, the variants IgG1-huCD3-H1L1 (humanized CD3         comprising the VH1 sequence set forth in SEQ ID NO:4 and the VL1         sequence set forth in SEQ ID NO:8), IgG1-huCD3-L1-T41K         (humanized CD3 comprising the VH1 sequence set forth in SEQ ID         NO:4 and the VL sequence set forth in SEQ ID NO:10.

b12 Antibody

-   -   In some of the examples the antibody b12, a HIV-1 gp120 specific         antibody (Barbas, C F. 3 Mol Biol. 1993 Apr. 5; 230(3):812-23.)         was used as a negative control, and is termed “IgG1-b12”.

Expression

-   -   Antibodies were expressed as IgG1,κ or IgG1,λ with or without         the non-activating mutations described below and with a mutation         in the CH3 domain enabling the generation of bispecific         antibodies by the method described below. Plasmid DNA mixtures         encoding both heavy and light chain of antibodies were         transiently transfected to Freestyle HEK293F cells (Invitrogen,         US) using 293fectin (Invitrogen, US) essentially as described by         the manufacturer.

Purification of Antibodies

-   -   Culture supernatant was filtered over 0.2 μm dead-end filters,         loaded on 5 mL MabSelect SuRe columns (GE Health Care) and         eluted with 0.1 M sodium citrate-NaOH, pH3. The eluate was         immediately neutralized with 2M Tris-HCl, pH9 and dialyzed         overnight to 12.6 mM NaH2PO4, 140 mM NaCl, pH7.4 (B.Braun).         Alternatively, subsequent to purification, the eluate was loaded         on a HiPrep Desalting column and the antibody was exchanged into         12.6 mM NaH2PO4, 140 mM NaCl, pH7.4 (B.Braun) buffer. After         dialysis or exchange of buffer, samples were sterile filtered         over 0.2 μm dead-end filters. Purity was determined by SDS-PAGE         and concentration was measured by absorbance at 280 nm. Purified         antibodies were stored at 2-8° C.

Example 2: Generation of Mutant Library

-   -   Point mutations were generated by random mutagenesis performed         using the Quick change mutagenesis kit (Stratagene, according to         the manufacturer's instructions and the HC (p33HGTE-huCD3-H1)         and LC (p33L-huCD3-L1-T41K) expression plasmids as templates.         The HC plasmid encodes for the monovalent UniBody—TE format as         described in WO2011110642. Each selected position was randomized         by using primers containing a NNS codon at the selected position         (N=G, A, T or C and S=G or C). Mutant libraries were transformed         to OneShot DH5alpha (Invitrogen) according to manufacturer's         instructions.

Colony Picking and LEE PCR

-   -   For each mutated position 96 clones were individually picked         into 50 μL LEE (linear expression element) PCR buffer (5 μL 10×         AccuPrime PCR buffer 1, 44.6 μL water (B.Braun), 0.1 μL CMV P f         (MARS) and 0.1 μL Tk pA r (MARI.) primers (100 μM stock), 0.2 μL         Accuprime Taq (Invitrogen) to amplify the expression cassette         from the expression plasmid (promoter up to poly A). LEE PCRs         were performed by incubating the mixtures 2′ 94° C., [30″ 94°         C., 30″ 55 OC, 5′ 68° C.]35x, 10′ 72° C. and storage at 4° C.         until further use.     -   Each library (12) of 96 colonies was sequenced using Sanger         sequencing (Beckman Coulter Genomics, UK).

TABLE 2 Primer sequences for LEE PCR Primer name Primer Sequence CMV P f GCTTCGCGATGTACGGGCCAGATATAC (MAR5) TK pA r GGATACCCCCTAGAGCCCCAGCTGCGCAGATCTGCT (MAR1) ATGGC

Example 3: Expression of Mutant Library and IgG Quantification

-   -   Of each mutant (12×96 in total) 1.11 μL HC and 1.11 μL LC LEE         PCR product were diluted in 2.78 μL water. The 5 μL DNA dilution         was used to transfect a single well in a 96 well plate.     -   Per well 0.4 μL ExpiFectamine™ 293 (Invitrogen, US) and 4.6 μL         Opti-MEM (Gibco, US) were mixed and incubated for 5 minutes at         room temperature. Next, the Fectin/Opti-MEM mix was added to the         5 μL DNA dilution and incubated for 30 minutes at room         temperature. Finally, 8.3 μL of the Fectin/Opti-MEM/DNA mix was         added to 117.5 μL Expi293F™ cells. During all procedures, the         plates with Expi293F™ cells were shaken to keep the cells in         suspension. After transfection, cells were incubated at 37°         C./8% CO₂ for 5 days.     -   Five days post transfection, the supernatant was harvested.         Antibody concentration in supernatant was measured by BioLayer         Interferometry using the Octet RED (ForteBio, US).

Example 4: Generation of CD3/TCR-LC13 Screening Library

Freestyle 293-F cells (Invitrogen, US) were co-transfected with expression constructs encoding the human alpha and beta chains of the TCR (SEQ ID NO: 396 and SEQ ID NO: 397 respectively), human CD3δ (SEQ ID NO: 398), human CD3ε (SEQ ID NO: 399), human CD3γ (SEQ ID NO: 400) and human CD3 (SEQ ID NO: 401). The signal peptide sequence is excluded in these sequences. Transfection was performed according to manufacturer's instructions (Invitrogen, US). One day post transfection, cells were frozen until further use.

Example 5: Screening of Affinity Mutants

Homogeneous Assay (Dose Response)

-   -   Based on the sequence data, mutants were selected where sequence         traces showed high PHRED scores, indicative for the absence of         multiple mutations. Per mutation, multiple redundant clones were         selected when available.     -   The binding of recombinantly produced UniBody molecules in cell         culture supernatant was determined by homogeneous antigen         specific binding assays using Fluorometric Micro volume Assay         Technology (FMAT; Applied Biosystems, Foster City, Calif., USA).         In the assay test design samples were analyzed in dose response         for binding of antibodies or monovalent antibody molecules to         CD3/TCR-LC13 (Freestyle 293-F cells transiently expressed human         CD3 and human T cell receptor (TCR); produced as described         above) and Freestyle 293-F wild-type cells (negative control         which does not express human TCR). IgG levels for sample         normalization prior to dose response binding were measured using         an Octet instrument (Fortebio, Menlo Park, USA).     -   Dilution series of samples were added to the cells to allow         binding to CD3. Subsequently, binding of monovalent antibody         molecules was detected using a fluorescent conjugate (Goat         anti-Human IgG Fc gamma-Alexa647; Jackson ImmunoResearch). The         CD3 specific humanized mouse antibody IgG1-HuM291-F405L         (produced in Freestyle 293-F cells) and monovalent anibody         UniTE-huCD3-H1L1-LT41K were used as a positive control and         ChromPure Human IgG, whole molecule (Jackson ImmunoResearch) was         used as negative control. The samples were scanned using an         Applied Biosystems 8200 Cellular Detection System (8200 CDS) and         total fluorescence over sample concentration was used as         read-out. Samples were stated positive when counts were higher         than 50 and counts x fluorescence (total florescence) was at         least three times higher than the negative control.

Heatmap

-   -   From the Homogeneous Dose Response screen, the binding curves         were fitted using a 4 parameter sigmoidal model. From the fit,         the maximal binding for every mutant was determined. Per mutant,         the average maximal binding was calculated, and depicted as a         ratio between average maximal over wt binding as shown in FIG. 1         .

Alignment

-   -   Selected HC mutants generated in these libraries are aligned and         depicted in FIG. 2 . The CDR regions have been annotated         according to the IMGT definitions. Numbering of the sequence is         annotated according to a direct numbering scheme.

Example 6—Generation of Bispecific Antibodies by 2-MEA-Induced Fab-Arm Exchange

The bispecific antibodies according to the invention may be generated by use of the methods disclosed in WO2011131746 and WO2013060867 (Genmab).

By way of example a mutation in position F405L may be introduced in one parental antibody of IgG1 isotype, and a mutation in position K409R may be introduced in the other parental antibody of IgG1 isotype.

These two parental antibodies, each at a final concentration of 0.5 mg/mL (equimolar concentration), may be incubated under reducing conditions with 25 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL Tris-EDTA (TE) at 37° C. for 90 min. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using spin columns (Microcon centrifugal filters, 30k, Millipore) according to the manufacturer's protocol.

The bispecific antibodies may be filtered over 0.2 μm dead-end filters and the absorbance at 280 nm (A280) of the bispecific antibodies may be measured to determine the final concentration thereof.

Example 7: Binding Data

-   -   For all binding assays below a selected panel of heavy chain         variants of huCD3-H1L1 were tested in different formats:

TABLE 3 Selected affinity variants of huCD3-H1L1 in monovalent antibody-TE format Selected UniTE-huCD3 HC affinity variants UniTE-huCD3-H1L1-LT41K (WT) UniTE-huCD3-H1L1-T31M-LT41K UniTE-huCD3-H1L1-T31P-LT41K UniTE-huCD3-H1L1-Y32A-LT41K UniTE-huCD3-H1L1-N57E-LT41K UniTE-huCD3-H1L1-H101F-LT41K UniTE-huCD3-H1L1-H101G-LT41K UniTE-huCD3-H1L1-H101I-LT41K UniTE-huCD3-H1L1-H101K-LT41K UniTE-huCD3-H1L1-H101L-LT41K UniTE-huCD3-H1L1-H101N-LT41K UniTE-huCD3-H1L1-G105P-LT41K UniTE-huCD3-H1L1-S110A-LT41K UniTE-huCD3-H1L1-S110G-LT41K UniTE-huCD3-H1L1-Y114M-LT41K UniTE-huCD3-H1L1-Y114R-LT41K UniTE-huCD3-H1L1-Y114V-LT41K

Octet Binding Affinity Determination of the CD3 Affinity Mutants in Monovalent Antibody-TE Format

-   -   Affinities of a selected panel of affinity VH variants (Table 3)         were determined using Bio-Layer Interferometry on a ForteBio         Octet HTX. Anti-human Fc Capture (AHC) biosensors (ForteBio,         Portsmouth, UK; cat no. 18-5060) were loaded for 600 s with the         CD3 affinity mutants in monovalent antibody-TE format (2 μg/mL),         aiming at a loading response of 0.4 nm. Antibodies of the         UniBody-TE format were used to specifically measure the         monovalent interaction affinity between the CD3 affinity mutants         and the CD3ε27-GSKa ligand. After a baseline (150 s) the         association (1000 s) and dissociation (1000 s) of CD3ε27-GSKa         (100 and 1000 nM) was determined. The CD3ε27-GSKa protein         consists of the human CD3ε peptide (aa1-27) fused to the         N-terminus of a kappa LC (SEQ ID NO: 402). For calculations, the         theoretical molecular mass of CD3ε27-GSKa based on the amino         acid sequence was used, i.e. 27.1 kDa. Experiments were carried         out while shaking at 1000 rpm and at 30° C.     -   Data was analyzed with ForteBio Data Analysis Software v8.1,         using the 1:1 model and a global full fit with 1000 s         association time and 200 s dissociation time. Data traces were         corrected by subtraction of a reference curve (CD3 affinity         mutant without CD3ε27-GSKa), the Y-axis was aligned to the last         5 s of the baseline, and interstep correction as well as         Savitzky-Golay filtering was applied.

TABLE 4 Equilibrium dissociation constant (KD) for selected variants Antibody ID KD (M) 1/KD (M⁻¹) UniTE-huCD3-H1L1-N57E-LT41K 2.4 × 10⁻⁸ 4.1 × 10⁷ uniTE-huCD3-H1L1-LT41K 3.4 × 10⁻⁸ 3.0 × 10⁷ UniTE-huCD3-H1L1-G105P-LT41K 3.5 × 10⁻⁸ 2.8 × 10⁷ UniTE-huCD3-H1L1-T31P-LT41K 4.9 × 10⁻⁸ 2.0 × 10⁷ UniTE-huCD3-H1L1-S110G-LT41K 6.1 × 10⁻⁸ 1.6 × 10⁷ UniTE-huCD3-H1L1-Y114V-LT41K 7.5 × 10⁻⁸ 1.3 × 10⁷ UniTE-huCD3-H1L1-Y114M-LT41K 7.6 × 10⁻⁸ 1.3 × 10⁷ UniTE-huCD3-H1L1-T31M-LT41K 2.0 × 10⁻⁷ 5.1 × 10⁶ UniTE-huCD3-H1L1-H101N-LT41K 2.0 × 10⁻⁷ 5.1 × 10⁶ UniTE-huCD3-H1L1-H101I-LT41K 2.2 × 10⁻⁷ 4.6 × 10⁶ UniTE-huCD3-H1L1-S110A-LT41K 3.2 × 10⁻⁷ 3.1 × 10⁶ UniTE-huCD3-H1L1-H101G-LT41K 9.7 × 10⁻⁷ 1.0 × 10⁶ UniTE-huCD3-H1L1-H101L-LT41K nd nd UniTE-huCD3-H1L1-Y32A-LT41K nd nd UniTE-huCD3-H1L1-H101K-LT41K nd nd UniTE-huCD3-H1L1-T31A-LT41K nd nd UniTE-huCD3-H1L1-H101F-LT41K nd nd UniTE-huCD3-H1L1-Y114R-LT41K nd nd nd = not determined

T Cell Binding of Affinity Variants of Humanized CD3 (UniTE-huCD3-H1L1-LT41K) on Flow Cytometry (FACS)

-   -   T cell binding of purified VH affinity variants of humanized CD3         (IgG1-huCD3-H1L1) antibodies was determined using         Fluorescence-Activated Cell Sorting on a FACSCanto 752 (BD         Biosciences). T cells were isolated from a buffy coat fraction         of anti-coagulated human donor blood samples and resuspended in         PBS/0.1% BSA/0.02% azide at 1.8×10E6 cells/mL. 50 μL of T cell         suspension and 50 μL of the antibody dilutions were combined in         a 96 well plate on ice, incubated for 30 min at 4° C. and washed         twice with PBS/0.1% BSA/0.02% azide. Next, 50 μL of secondary         antibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG         F(ab′)2 (109-116-098, Jackson ImmunoResearch Laboratories, Inc.,         West Grove, Pa.) diluted 1/200 in PBS/0.1% BSA/0.02% azide, was         added for staining, the mixture was incubated for 30 min at         4° C. and subsequently washed twice with PBS/0.1% BSA/0.02%         azide. The cells were resuspended in 120 μL PBS/0.1% BSA/0.02%         azide and PE geometric Mean Fluorescence Intensity was measured.         Binding curves were analyzed using non-linear regression         (sigmoidal dose-response with variable slope) using GraphPad         Prism V5.04 software (GraphPad Software, San Diego, Calif., USA)         and apparent affinity (K_(D)) was derived from the concentration         at half-maximal binding. FIG. 4 shows binding curves of affinity         variants of humanized CD3 (UniTE-huCD3-H1L1-LT41K) and FIG. 5         shows the binding curves of low affinity variants of humanized         CD3 (UniTE-huCD3-H1L1-LT41K).

TABLE 5 Summary of binding data of CD3 affinity mutants in monovalent antibody-TE format Tcell binding average at Max 5000 ng/ml CD3/ (Geometric TCRLC13 KD antibody ID MFI) (RFU) (M) UniTE-huCD3-H1L1- 13206 2030549 3.4 × 10⁻⁸ LT41K UniTE-huCD3-H1L1- 10526 2628228 3.5 × 10⁻⁶ G105P-LT41K UniTE-huCD3-H1L1- 10240 1836187 T31A-LT41K UniTE-huCD3-H1L1- 8492 1788851 2.4 × 10⁻⁸ N57E-LT41K UniTE-huCD3-H1L1- 6197 1599412 Y114R-LT41K UniTE-huCD3-H1L1- 6025 1807485 7.5 × 10⁻⁸ Y114V-LT41K UniTE-huCD3-H1L1- 4286 1718317 4.9 × 10⁻⁸ T31P-LT41K UniTE-huCD3-H1L1- 3830 1771714 7.6 × 10⁻⁸ Y114M-LT41K UniTE-huCD3-H1L1- 1919 1539506 2.0 × 10⁻⁷ T31M-LT41K UniTE-huCD3-H1L1- 1660 1411016 3.2 × 10⁻⁷ S110A-LT41K UniTE-huCD3-H1L1- 1321 893765 H101K-LT41K UniTE-huCD3-H1L1- 949 385873 6.1 × 10⁻⁸ S110G-LT41K UniTE-huCD3-H1L1- 848 1060228 2.0 × 10⁻⁷ H101N-LT41K UniTE-huCD3-H1L1- 719 541497 Y32A-LT41K UniTE-huCD3-H1L1- 641 H101F-LT41K UniTE-huCD3-H1L1- 511 603683 2.2 × 10⁻⁷ H101I-LT41K UniTE-huCD3-H1L1- 509 812768 H101L-LT41K UniTE-huCD3-H1L1- 492 139712 9.7 × 10⁻⁷ H101G-LT41K

For all binding assays below a selected panel of preferred heavy chain variants was tested (see Table 5)

Octet Binding Affinity Determination of IgG1-huCD3-H1L1-FEAL Affinity Mutants

Affinities of selected CD3 affinity variants in an IgG1-huCD3-H1L1-FEAL format were determined using Bio-Layer Interferometry on a ForteBio Octet HTX (ForteBio, UK) (Table 6). Anti-human Fc capture biosensors (cat: 18-5060, ForteBio, UK) were loaded for 600 s with hIgG (1 μg/mL). After a baseline (200 s) the association (1000 s) and dissociation (2000 s) of CD3ε27-GSKa was determined, using a CD3ε27-GSKa concentration range of 27.11 μg/mL-0.04 μg mL (1000 nM-1.4 nM) with three-fold dilution steps (sample diluent, cat: 18-5028, ForteBio, UK). For calculations, the theoretical molecular mass of CD3ε27-GSKa based on the amino acid sequence was used, i.e. 27.11 kDa. Experiments were carried out while shaking at 1000 rpm and at 30° C. Each antibody was tested in at at least two independent experiments (Table 6).

Data was analyzed with ForteBio Data Analysis Software v8.1, using the 1:1 model and a global full fit with 1000 s association time and 100 s dissociation time. Data traces were corrected by subtraction of a reference curve (antibody without CD3ε27-GSKa), the Y-axis was aligned to the last 10 s of the baseline, and interstep correction as well as Savitzky-Golay filtering was applied. Data traces with a response <0.05 nm were excluded from analysis.

TABLE 6 <KD> Average (nM) SDEV SEM CV <Kon> SDEV SEM CV <Kdis> SDEV SEM CV pKD n IgG1-huCD3-G105P-FEAL 5 2 1 45 4.7E+05 9.7E+04 5.6E+04 21 2.5E−03 1.0E−03 5.8E−04 40 8.3 3 IgG1-huCD3-FEAL 15 6 3 37 2.7E+05 5.1E+04 2.9E+04 19 4.0E−03 1.6E−03 9.1E−04 39 7.8 3 IgG1-huCD3-Y114V-FEAL 29 8 4 26 2.2E+05 3.3E+04 1.9E+04 15 6.3E−03 9.7E−04 5.6E−04 15 7.5 3 IgG1-huCD3-T31P-FEAL 42 9 4 21 1.9E+05 3.8E+04 1.6E+04 20 7.8E−03 1.3E−03 5.3E−04 17 7.4 6 IgG1-huCD3-Y114M-FEAL 42 14 8 33 2.6E+05 6.2E+04 3.6E+04 24 1.0E−02 1.5E−03 8.7E−04 15 7.4 3 IgG1-huCD3-H101N-FEAL 45 13 7 29 4.8E+05 2.2E+05 1.2E+05 45 2.0E−02 3.1E−03 1.8E−03 16 7.3 3 IgG1-huCD3-Y114R-FEAL 46 10 6 22 1.5E+05 4.1E+04 2.4E+04 27 6.8E−03 4.1E−04 2.4E−04 6 7.3 3 IgG1-huCD3-S110A-FEAL 72 15 6 21 1.8E+05 2.5E+04 1.0E+04 14 1.3E−02 1.6E−03 6.4E−04 12 7.1 6 IgG1-huCD3-N57E-FEAL 91 30 17 33 2.1E+05 2.8E+04 1.6E+04 13 1.9E−02 4.0E−03 2.3E−03 21 7.0 3 IgG1-huCD3-T31M-FEAL 99 23 13 23 1.9E+05 2.5E+04 1.5E+04 14 1.8E−02 2.6E−03 1.5E−03 14 7.0 3 IgG1-huCD3-Y32A-FEAL 105 31 22 29 2.2E+05 1.1E+05 7.5E+04 48 2.2E−02 4.4E−03 3.1E−03 20 7.0 2 IgG1-huCD3-H101L-FEAL 107 39 23 37 2.7E+05 4.3E+04 2.5E+04 16 2.8E−02 7.5E−03 4.4E−03 27 7.0 3 IgG1-huCD3-H101K-FEAL 120 94 55 79 2.2E+05 1.9E+05 1.1E+05 84 1.7E−02 9.8E−03 5.7E−03 58 6.9 3 IgG1-huCD3-S110G-FEAL 153 120 70 79 3.8E+05 4.2E+05 2.4E+05 112 2.6E−02 8.3E−03 4.8E−03 32 6.8 3 IgG1-huCD3-H101G-FEAL 683 169 97 25 3.0E+04 9.2E+03 5.3E+03 30 2.0E−02 8.5E−04 4.9E−04 4 6.2 3 IgG1-huCD3-H101I-FEAL nd 0 IgG1-huCD3-H101F-FEAL nd 0

T Cell Binding Affinity Determination of IgG1-huCD3-H1L1-FEAL Affinity Mutants

T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. Briefly, 50 μL of T cell isolation cocktail was added to 1 mL of buffy coat and incubate at RT for 20 min. Next, the buffy coat was diluted (1:3, v/v) with PBS (cat: 3623140, B.Braun, Germany) and gently transferred to 50 mL falcon tubes (cat: 227261, Greiner bio-one, The Netherlands) filled with 15 mL lymphocyte separation medium (cat: 17-829E, Lonza, Switzerland). Tubes were centrifuged for 20 min at RT 1200×g without brakes. Collect the T cells from the density medium and wash with PBS twice.

2×10E6 T cells/mL were resuspended in FACS buffer and transferred to 50 μL into round bottom 96 well plates (cat: 650101, Greiner bio-one, The Netherlands). 50 μl of the antibody solutions in five-fold dilutions was added starting with 5 μg/mL and incubated for 30 min at 4° C. The 96 plates were centrifuged at 300×g for 5 min at 4° C. and the supernatant discarded. Cells were washed twice with ice cold FACS buffer on ice and the 1:200 diluted secondary antibody (anti IgG Fcγ-PE (fab)′2, cat: 109-116-098, Jackson Immuno Research, UK) added to 100 4/well and incubated for 30 min and washed twice with FACS buffer. Fluorescence intensity was measured on FACS Canto and geometric mean calculated by FlowJo V10 software. Graphs were made by GraphPad (V6.04). See FIG. 5 .

Example 9: In Vitro Cytotoxicity Screening of CD3 Affinity Mutants

Cytotoxicity of CD3 Affinity Mutants on Solid Tumor Cell Lines (Alamar Blue Assay)

T cells from donor buffy coats (Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. NCI-N87 (25.000 cells/well) (FIG. 6A), SKOV3 (16.000 cells/well) (FIG. 6B) and MDA-MB-231 (16.000 cells/well) (FIG. 6C) cells were seeded into flat bottom 96 well plates (cat: 655180, Greiner-bio-one, The Netherlands) and adhered for 3-5h at 37° C. T cells were added to tumor cells in the following ratios: NCI-N87 cell: T cells, 1:3; SKOV3 cell:T cell, 1:4; MDA-MB-231 cell: T cell, 1:8. Subsequently antibody solutions were added in ten-fold dilutions and plates were incubated for 2 days at 37° C. Next, supernatants were discarded and adhered cells were washed twice with PBS. 150 μL of 10% alamar blue (cat: DAL1100, Life Technologies, The Netherlands) solution prepared in RPMI-1640 (cat: BE12-115F, Lonza, Switzerland) medium containing 10% donor bovine serum with iron (cat: 10371-029, Life Technologies, The Netherlands) was added to wells and incubated for 3-5h at 37° C. The absorbance was measured with Envision multilabel plate reader (PerkinElmer, US). Staurosporine (cat: S6942, Sigma-Aldrich, US) treated cells were set as 100% kill and untreated cells were set as 0% kill. Viable cells were calculated by subtracting staurosporine treated cells from all groups and the percentage was plotted against the untreated group. Graphs were made by GraphPad (V6.04). See FIG. 6 .

Cytotoxicity of CD3 Affinity Mutants on a Hematological Cell Line (Chromium Release Assay)

5×10E6 Daudi cells/mL were incubated in complete culture medium with 100 μCi chromium for 1h under shaking conditions at 37° C. Next, cells were washed twice in PBS and resuspended in 5 mL complete cell culture medium (10% donor bovine serum with iron in RPMI 1640). 5.000 Daudi cells were seeded into round bottom 96 well plates. T cells from donor buffy coats (purchased from Sanquin, Amsterdam, The Netherlands) were isolated by using RosetteSep human T cell enrichment cocktail (Cat: 15021C.1, Stemcell Technologies, France) according to manufacturer's instructions. T cells are added in (tumor cell: T cell) 1:10 ratio to Daudi cells followed by the addition of the antibody solutions in two-fold dilutions. Plates were in incubated 24h at 37° C. After 24h, plates were centrifuges at 300×g for 3 min, supernatant was harvested and measured for radioactivity. See FIG. 7 .

TABLE 7 % of kill HER2  

  Cells No kill kill  

  kill  

  kill  

  HER2  

  NC 

 - H101 

  H101 

   

   

 110 

  N 

  H101 

   

  H101 

  G 

  H101 

  H101 

  S110A, Y32 

   

 M,  

 P, Y114M, Y114 

  Y114V 50 

 000 

   

  H101 

  H101 

  H101 

   

 110 

   

 000 H101 

   

  G 

  H101 

  H101 

  S110A, Y32 

   

   

 M,  

 P, Y114M, Y114 

  Y114V HER2  

 000  

 - H101 

  H101 

   

 110 

  M 

 - H101 

  G 

   

   

  S110A, Y32 

   

 M, H101 

   

 P, H101 

  Y114M, H101 

  Y114 

   

  Y114V

indicates data missing or illegible when filed

Example 10: Tumor Efficacy of CD3×HER2 Bispecific Antibodies in a, (Human PBMC+NCI-N87 Cells) Co-Engraftment Model in NOD-SCID Mice

The in vivo anti-tumor efficacy of several CD3×HER2 bispecific antibodies was evaluated in a subcutaneous NCI-N87 co-engraftment model (FIG. 8 ). As a CD3-arm of the bispecific antibody, a humanized WT CD3 (huCD3-FEAL) and 4 different CD3 affinity variants (N57E, H101K, S110A, Y114M) were used. The HER2-targeting arm in all cases was the same (Herceptin-FEAR)

BisG1-h u CD3-FEALx1014-Herceptin-FEAR

BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR

BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR

BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR

BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR

In this model, HLA-A-matched human unstimulated PBMCs, as a source of human T cells, were co-inoculated with NCI-N87 tumor cells at two different dose levels (0.5 and 0.05 mg/kg).

Mice were sorted into treatment groups (n=4 per treatment group. At day 0, a mixture containing HLA-A matched hPBMC (5×10E6, Sanquin) and NCI-N87 (5×10E6) cells in 200 μL PBS/0.1% BSA were inoculated subcutaneously (s.c.) in the right flank of each female NOD-SCID mice (NOD.C.B-17-Prkdc^(scid)/J), age 6-11 weeks old (Charles-River)). Directly after tumor cell injection, single intravenous dosing (150 4) of five different CD3×HER2 antibodies was performed at two different concentrations (0.5 and 0.05 mg/kg) for all bispecific antibodies. Tumor volumes were determined at least two times per week. Tumor volumes (mm³) were calculated from caliper (PLEXX) measurements as: 0.52×(length)×(width)².

NCI-N87 cells (ATCC # CRL-5822, gastric carcinoma arising from stomach) were thawed, cultured in RPMI 1640 (Lonza, BE12-115F) supplemented with 10% donor bovine serum with iron (Gibco, cat. no. 10371-029), penicillin/streptomycin and 0.45% glucose (Sigma, G8769), sodium pyruvate (Cambrex, 13E13-115E) and 0.075% sodium bicarbonate (Cambrex, BE17-613E). Cells were grown in CelISTACK culture chambers and harvested in log-phase and counted by trypan blue exclusion.

For each study, hPBMCs were isolated from human HLA-A matched donors for NCI-N87 (HLA-A-01,23) from a buffy coat (Sanquin) by Ficoll density centrifugation. Isolated cells were frozen in nitrogen and thawed before use. All cells were washed in PBS/0.1% BSA, filtered through a cell strainer and resuspended to a concentration of 50×10E6 cells/mL in PBS/0.1% BSA.

The results are shown in FIG. 8 . FIG. 6A-B show average tumor volumes after treatment over time. FIG. 6C-D show dot plot representations of average NCI-N87 tumor volume at day 44. Statistical analysis (Mann-Whitney) on the tumor volumes at day 44, last day that all groups were still intact, revealed at a dose of 0.05 mg/kg a significant tumor growth inhibition (p<0.05) of BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR compared to the control (PBMCs) and not of BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR and BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR. At a dose of 0.5 mg/kg BisG1-huCD3-FEALx1014-Herceptin-FEAR and all CD3-arm affinity variants, showed significant tumor growth (p<0.05) inhibition compared to the PBS (PBMCs) control group, except BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR.

BisG1-huCD3-FEALx1014-Herceptin-FEAR, BisG1-huCD3-S110A-FEALx1014-Herceptin-FEAR, and BisG1-huCD3-Y114M-FEALx1014-Herceptin-FEAR significantly (p<0.05) reduced NCI-N87 tumor volume at dosages of 0.05 and 0.5 mg/kg. BisG1-huCD3-N57E-FEALx1014-Herceptin-FEAR reduced significantly (p<0.05) NCI-N87 tumor volume only at a dosage of 0.5 mg/kg. BisG1-huCD3-H101K-FEALx1014-Herceptin-FEAR did not affect NCI-N87 tumor growth at both tested dosages.

LIST OF REFERENCES

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1-77. (canceled)
 78. A method of treating cancer, an infectious disease, or an autoimmune disease, the method comprising administering to a subject in need thereof an effective amount of a chimeric or humanized antibody which binds to human CD3, a bispecific antibody comprising the antigen binding region of the antibody, or a composition comprising the antibody or bispecific antibody and a carrier, wherein the antibody comprises a heavy chain variable (VH) region, wherein said VH region comprises a mutation in one of the three CDR sequences of a reference antibody having the CDR sequences set forth in SEQ ID NOs: 1, 2, and 3, which mutation is in one of the positions selected from the group consisting of: T31M, T31P, N57, H101, G105, S110 and Y114, wherein the positions are numbered according to the reference sequence of SEQ ID NO:
 4. 79. The method according to claim 78, wherein the antibody comprises an H101G or an H101N mutation.
 80. The method according to claim 78, wherein the VH region of the antibody comprises the CDR1, CDR2, and CDR3 sequences selected from the group consisting of: a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2, 3 [T31M]; b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2, 3 [T31P]; c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106, 3 [N57E]; d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 176 [H101G]; e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 184 [H101N]; f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 220 [G105P]; g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 236 [S110A]; h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 244 [S110G]; i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 284 [Y114M]; j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 292 [Y114R]; k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, 298 [Y114V]; and l) CDR1, CDR2 and CDR3 sequences having at least 90% or at least 95% amino acid sequence identity, in total across the three CDR sequences or to any one of the three CDR sequences as set forth in a) to k), or at most 5 further mutations or substitutions in total across the three CDR sequences, provided that the CDR1, CDR2 and CDR3 sequences do not have the sequences as set forth in SEQ ID NO: 1, 2, 3, and wherein the mutations or substitutions preferably do not modify the binding affinity of the antibody to human CD3.
 81. The method according to claim 78, wherein the VH region of the antibody comprises an amino acid sequence selected from the group consisting of: a) a VH sequence as set forth in SEQ ID NO: 55[T31M], b) a VH sequence as set forth in SEQ ID NO: 59 [T31P], c) a VH sequence as set forth in SEQ ID NO: 107 [N57E] d) a VH sequence as set forth in SEQ ID NO: 177 [H101G], e) a VH sequence as set forth in SEQ ID NO: 185 [H101N], f) a VH sequence as set forth in SEQ ID NO: 221 [G105P], g) a VH sequence as set forth in SEQ ID NO: 237 [S110A], h) a VH sequence as set forth in SEQ ID NO: 245 [S110G], i) a VH sequence as set forth in SEQ ID NO: 285 [Y114M], j) a VH sequence as set forth in SEQ ID NO: 293 [Y114R], k) a VH sequence as set forth in SEQ ID NO: 299 [Y114V], and l) a VH sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to any one of the sequences as set forth in a) to k).
 82. The method according to claim 78, wherein the antibody comprises a light chain variable region comprising CDR1, CDR2, and CDR3 sequences set forth in SEQ ID NO:6, GTN, and 7, respectively.
 83. The method according to claim 78, wherein the antibody comprises a variable light chain (VL) region, wherein said VL region is selected from the group consisting of: a) a VL sequence as set forth in SEQ ID NO:8, b) a VL sequence as set forth in SEQ ID NO:10, and c) a VL sequence having at least 90%, at least 95%, at least 97% or at least 99% amino acid sequence identity to any one of the sequences as set forth in a) to b).
 84. The method according to claim 82, wherein the antibody comprises a VH region comprising CDR1, CDR2, and CDR3 sequences selected from the group consisting of: a) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 54, 2, and 3 [T31M]; b) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 58, 2, and 3 [T31P]; c) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 106, and 3 [N57E]; d) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 176 [H101G]; e) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 184 [H101N]; f) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 220 [G105P]; g) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 236 [S110A]; h) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 244 [S110G]; i) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 284 [Y114M]; j) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 292 [Y114R]; and k) CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO: 1, 2, and 298 [Y114V].
 85. The method according to claim 78, wherein the antibody is a full-length antibody.
 86. The method according to claim 78, wherein the antibody is chimeric or humanized.
 87. The method according to claim 78, wherein the antibody is of an isotype selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.
 88. The method according to claim 78, wherein the antibody is monovalent, bivalent or multivalent.
 89. The method according to claim 78, wherein the antibody comprises a first heavy chain and a second heavy chain.
 90. The method according to claim 78, wherein the antibody comprises a first heavy chain and a second heavy chain, wherein in at least one of said first and second heavy chains, one or more amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering, are not L, L, D, N, and P, respectively.
 91. The method according to claim 89, wherein in at least one of said first and second heavy chains of the antibody, (a) the amino acid in the position corresponding to position D265 in a human IgG1 heavy chain is not D; (b) the amino acid in the position corresponding to position N297 in a human IgG1 heavy chain is not N; (c) the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively; (d) the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E; or A and A, respectively; (e) the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively; (f) the amino acids in the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are A and A, respectively; (g) the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are not L, L, and D, respectively; (h) the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A; or A, A, and A, respectively; (i) the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively; (j) the amino acids in the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain, are A, A, and A, respectively; or (k) the amino acids in the positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are F, E, A, Q, and S, respectively.
 92. The method according to claim 89, wherein each of said first and second heavy chain of the antibody comprises at least a hinge region, a CH2 region, and a CH3 region, wherein in said first heavy chain, at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second heavy chain, at least one of the amino acids in the positions corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein said first and said second heavy chains are not substituted in the same positions.
 93. The method according to claim 92, wherein (a) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said first heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said second heavy chain, or (b) the amino acid in the position corresponding to F405 in a human IgG1 heavy chain is L in said second heavy chain, and the amino acid in the position corresponding to K409 in a human IgG1 heavy chain is R in said first heavy chain.
 94. The method according to claim 78, wherein the subject has cancer.
 95. The method according to claim 94, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, non-small cell lung cancer, bladder cancer, ovarian cancer, gastric cancer, colorectal cancer, esophageal cancer and squamous cell carcinoma of the head & neck, cervical cancer, pancreatic cancer, testis cancer, malignant melanoma, a soft-tissue cancer, an indolent or aggressive form of B-cell lymphoma, chronic lymphatic leukemia, and acute lymphatic leukemia.
 96. The method according to claim 78, wherein the subject has an infectious disease.
 97. The method according to claim 78, wherein the subject has an autoimmune disease. 